Reagents and methods for using human embryonic stem cells to evaluate toxicity of pharmaceutical compounds and other chemicals

ABSTRACT

The invention provides biomarker profiles of cellular metabolites and methods for screening chemical compounds including pharmaceutical agents, lead and candidate drug compounds and other chemicals using human embryonic stem cells (hESC) or lineage-specific cells produced therefrom. The inventive methods are useful for testing toxicity, particularly developmental toxicity and detecting teratogenic effects of such chemical compounds.

This is a divisional of U.S. application Ser. No. 11/733,677 filed onApr. 10, 2007 which application claims the priority benefit of U.S.provisional patent applications, Ser. Nos. 60/790,647, filed Apr. 10,2006, and 60/822,163, filed Aug. 11, 2006, the entirety of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention provides methods for toxicological screening ofpharmaceuticals and other chemical compounds. The invention specificallyprovides reagents that are human embryonic stem cells (hESC) orhESC-derived lineage-specific cells, such as neural stem cells, neuralprecursor cells and neural cells, as well as methods for using thesecells to detect developmental toxicity or teratogenic effects ofpharmaceutical compounds and other chemicals. More particularly, theinvention provides an in vitro means for analyzing toxicity of compoundspredictive of their toxicity during human development. Candidatepredictive biomarkers for toxic or teratogenic effects are alsoidentified and provided herein.

2. Background of Invention

Birth defects are a major cause of infant morbidity in the UnitedStates, affecting 1 in every 33 infants born (Brent & Beckman, 1990,Bull NY Acad Med 66: 123-63; Rosano et al., 2000, J EpidemiologyCommunity Health 54:660-66), or approximately 125,000 newborns per year.It is understood that developmental toxicity can cause birth defects,and can generate embryonic lethality, intrauterine growth restriction(IUGR), dysmorphogenesis (such as skeletal malformations), andfunctional toxicity, which can lead to cognitive disorders such asautism. There is an increasing concern about the role that chemicalexposure can play in the onset of these disorders. Indeed, it isestimated that 5% to 10% of all birth defects are caused by in uteroexposure to known teratogenic agents (Beckman & Brent, 1984, Annu RevPharmacol 24: 483-500).

Concern exists that chemical exposure may be playing a significant andpreventable role in producing birth defects (Claudio et al., 2001,Environm Health Perspect 109: A254-A261). This concern has beendifficult to evaluate, however, since the art has lacked a robust andefficient model for testing developmental toxicity for the more than80,000 chemicals in the market, plus the new 2,000 compounds introducedannually (General Accounting Office (GAO), 1994, Toxic SubstancesControl Act: Preliminary Observations on Legislative Changes to MakeTSCA More Effective, Testimony, 07/13/94, GAO/T-RCED-94-263). Fewer than5% of these compounds have been tested for reproductive outcomes andeven fewer for developmental toxicity (Environmental Protective Agency(EPA), 1998, Chemical Hazard Data Availability Study, Office ofPollution Prevention and Toxins). Although some attempts have been madeto use animal model systems to assess toxicity (Piersma, 2004,Toxicology Letters 149:147-53), inherent differences in the sensitivityof humans in utero have limited the predictive usefulness of suchmodels. Development of a human-based cell model system would have anenormous impact in drug development and risk assessment of chemicals.

Toxicity, particularly developmental toxicity, is also a major obstaclein the progression of compounds through the drug development process.Currently, toxicity testing is conducted on animal models as a means topredict adverse effects of compound exposure, particularly ondevelopment and organogenesis in human embryos and fetuses. The mostprevalent models that contribute to FDA approval of investigational newdrugs are whole animal studies in rabbits and rats (Piersma, 2004,Toxicology Letters 149:147-53). In vivo studies rely on administrationof compounds to pregnant animals at different stages of pregnancy andembryonic/fetal development (first week of gestation, organogenesisstage and full gestation length). However, these in vivo animal modelsare limited by a lack of robustness between animal and human responsesto chemical compounds during development. Species differences are oftenmanifested in trends such as dose sensitivity and pharmacokineticprocessing of compounds. At present, animal models are only 50%efficient in predicting human developmental response to compounds(Greaves et al., 2004, Nat Rev Drug Discov 3:226-36). Thus,human-directed predictive in vitro models present an opportunity toreduce the costs of new drug development and enable safer drugs.

In vitro models have been employed in the drug industry for over 20years (Huuskonen, 2005, Toxicology & Applied Pharm 207:S495-S500). Manyof the current in vitro assays involve differentiation models usingprimary cell cultures or immortalized cells lines (Huuskonen, 2005,Toxicology & Applied Pharm 207:5495-S500). Unfortunately, these modelsdiffer significantly from their in vivo counterparts in their ability toaccurately assess development toxicity. In particular, the ECVAMinitiative (European Center for Validation of Alternative Methods) hasused mouse embryonic stem cells as a screening system for predictivedevelopmental toxicology. The embryonic stem cell test (EST) has shownvery promising results, with a 78% statistically significant correlationto in vivo studies, and the test was able to differentiate strongteratogens from moderate/weak or non-embryotoxic compounds (Spielmann etal., 1997, In Vitro Toxicology 10:119-27). This model is limited in partbecause toxicological endpoints are defined only for compounds thatimpair cardiac differentiation. This model also fails to account forinterspecies developmental differences between mice and humans, and sodoes not fully address the need in the art for human-specific modelsystems.

Thus there remains a need in this art for a human-specific in vitromethod for reliably determining developmental toxicity in pharmaceuticalagents and other chemical compounds. There also is a need in the art tobetter understand human development and its perturbation by toxins andother developmental disrupting agents, to assist clinical management ofacquired congenital disorders and the many diseases that share thesebiochemical pathways, such as cancer.

The present invention provides for the assessment of a plurality ofsmall molecules, preferably secreted or excreted by hES cells orhESC-derived lineage-specific cells, such as neural stem cells, neuralprecursor cells and neural cells, and is determined and correlated withhealth and disease or insult state. Similar analyses have been appliedto other biological systems in the art (Want et al., 2005 Chem Bio Chem6: 1941-51), providing biomarkers of disease or toxic responses that canbe detected in biological fluids (Sabatine et al., 2005 Circulation112:3868-875).

SUMMARY OF THE INVENTION

The present invention provides reagents and methods for in vitroscreening of toxicity and teratogenicity of pharmaceutical andnon-pharmaceutical chemicals using undifferentiated human embryonic stemcells (hESC) or hESC-derived lineage-specific cells, such as neural stemcells, neural precursor cells and neural cells. The invention provideshuman-specific in vitro methods for reliably determining toxicity,particularly developmental toxicity and teratogenicity, ofpharmaceuticals and other chemical compounds using human embryonic stemcells (hESCs) or hESC-derived lineage-specific cells, such as neuralstem cells, neural precursor cells and neural cells. As provided herein,hESCs or hESC-derived lineage-specific cells, such as neural stem cells,neural precursor cells and neural cells, are useful for assessing toxiceffects of chemical compounds, particularly said toxic and teratogeniceffects on human development, thus overcoming the limitations associatedwith interspecies animal models. In particular, the inventiondemonstrates that metabolite profiles of hES cells or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells are altered in response to known disruptors ofhuman development.

The invention shows that the hESC metabolome is a source of humanbiomarkers for disease and toxic response. In particular embodiments,exposure of hESC to valproate induced significant changes in differentmetabolic pathways, consistent with its known activity as a humanteratogen. In other embodiments, hESC exposure to varying levels ofethanol induced significant alterations in metabolic pathways consistentwith alcohol's known effects on fetal development.

In one aspect, the invention provides methods for using undifferentiatedpluripotent human embryonic stem cells (hESC) or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells, for in vitro evaluation. In the inventivemethods, undifferentiated hESCs or hESC-derived lineage-specific cells,such as neural stem cells, neural precursor cells and neural cells areexposed to test compounds, preferably at concentrations reflective of invivo levels or at levels found in maternal circulation. Furtherembodiments of this aspect of the invention provide for determination ofthe capacity of the test compound to induce differentiation ofpluripotent hESC into particular cell types. In other embodiments, theinventive methods are provided using pluripotent, non-lineage restrictedcells. The benefit of utilizing pluripotent stem cells is they permitanalysis of global toxic response(s) and are isolated from thephysiological target of developmental toxicity, i.e. the human embryo.In addition, because these cells have not differentiated into a specificlineage, the potential for false negatives is reduced. In yet furtherembodiments are provided methods using hESC-derived lineage-specificcells, such as neural stem cells, neural precursor cells and neuralcells, for assessing toxicity and particularly developmental toxicityand teratogenicity.

In another aspect the invention provides methods for identifyingpredictive biomarkers of toxic responses to chemical compounds,particularly pharmaceutical and non-pharmaceutical chemicals, andparticularly to known teratogens. In embodiments of this aspect, adynamic set representative of a plurality of cellular metabolites,preferably secreted or excreted by hES cells or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells, is determined and correlated with health anddisease or toxic insult state. Cellular metabolites according to thisaspect of the invention generally range from about 10 to about 1500Daltons, more particularly from about 100 to about 1000 Daltons, andinclude but are not limited to compounds such as sugars, organic acids,amino acids, fatty acids and signaling low-molecular weight compounds.Said biomarker profiles are diagnostic for toxicity of chemicalcompounds, particularly pharmaceutical and non-pharmaceutical chemicals,that participate in and reveal functional mechanisms of cellularresponse to pathological or toxic chemical insult, thus serving asbiomarkers of disease or toxic response that can be detected inbiological fluids. In particularly preferred embodiments of this aspectof the invention, these biomarkers are useful for identifying active (oractivated) metabolic pathways following molecular changes predicted,inter alia, by other methods (such as transcriptomics and proteomics).

The invention thus also provides biomarker and pluralities ofbiomarkers, in some instances associated with metabolites fromparticular metabolic pathways, that are indicative of toxic orteratogenic insult. Said markers as provided by the invention are usedto identify toxic and teratogenic insult, and in particular embodimentsare used to characterize the amount or extent of said insult by beingcorrelated with the amount or extent of the particular biomarker orplurality of biomarkers detected in cell culture media. In particularembodiments, said plurality of biomarkers provide a diagnostic patternof toxic or teratogenic insult, more particularly identifying one or amultiplicity of specific metabolic pathways comprising metabolitesdetected after toxic or teratogenic insult.

The present invention is advantageous compared with inter alia the ECVAMmouse model because toxicity testing and biomarker identification areperformed with human cells, specifically human embryonic stem cells(hESC). Human embryonic stem cells are able to recapitulate mammalianorganogenesis in vitro (Reubinoff et al., 2000, Nature Biotechnology18:399-404; He et al., 2003, Circ Res 93:32-9; Zeng et al., 2004, StemCells 22:925-40; Lee et al., 2000, Mol Genet Metab 86:257-68; Yan etal., 2005, Stem Cells 22:781-90) because they are pluripotent andself-renewing cells. Thus, hESCs can reveal mechanisms of toxicity,particularly developmental toxicity, and identify developmental pathwaysthat are particularly sensitive to chemicals during early humandevelopment. The “human for human” embryonic model provided by theinventive methods disclosed herein permits a better understanding of thepathways associated with developmental toxicity, as this is a systemdeveloped directly from the target organism, as well as being a moreaccurate and sensitive assay for toxic or teratogenic insult in humandevelopment.

The methods of the invention provide further advantages in identifyingimportant biomarkers for toxicity and teratogenicity by functionalscreening of hESCs or hESC-derived lineage-specific cells, such asneural stem cells, neural precursor cells and neural cells. Thesebiomarkers advantageously identify metabolic and cellular pathways andmechanisms of toxicity, particularly developmental toxicity.Importantly, these biomarkers may also assist in the evaluation of toxiceffects of chemicals on the developing human embryo.

In yet another aspect of the invention, differentially-detected secretedor excreted cellular products identified by methods of the inventioninclude those associated with neurodevelopmental disorders andalterations in associated metabolic pathways, and include but are notlimited to kynurenine, glutamate, pyroglutamic acid,8-methoxykynurenate, N′-formylkynurenine 5-hydroxytryptophan,N-acetyl-D-tryptophan and other metabolites in the tryptophan andglutamate metabolic pathways.

Functional toxicity in post-natal life can be predicted using hESC sincedifferentiated cells with critical in vivo properties can be generatedin vitro. hESCs can be used to produce lineage-specific cells, includinglineage-specific stem cells, precursor cells andterminally-differentiated cells, providing therein enriched populationsof cells typically present in vivo in mixtures of different cell typescomprising tissues. The invention thus provides methods for using hESCsto produce said enriched and developmental stage-specific populations ofcells for toxicity screening of chemical compounds, particularly drugs,drug lead compounds and candidate compounds in drug development, toidentify human-specific toxicities of said chemical compounds. Theseaspects of the methods of the invention are advantageous overart-recognized in vitro and in vivo animal model systems.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects and features of this invention will be betterunderstood from the following detailed description taken in conjunctionwith the drawing wherein:

FIGS. 1A through 1C are profiles of secreted cellular metabolitebiomarkers produced after contacting hESCs with 1 mM valproate. Theseprofiles were produced using liquid chromatography/electrosprayionization-time of flight (TOF) mass spectrometry (LC/ESI-TOF-MS) aftertreating the cells with valproate for 24 hours (FIG. 1A), four days(FIG. 1B) and eight days (FIG. 1C). Secreted small molecules fromtreated (blue) and untreated (red) human embryonic stem cells weremeasured.

FIGS. 2A through 2D are profiles of secreted/excreted cellularmetabolite biomarkers produced after contacting hESCs with 1 mMvalproate. These profiles were produced using liquidchromatography/electrospray ionization time of flight mass spectrometry(LC/ESI-TOF-MS) after treating cells with valproate for 24 hours (FIG.2A), four days (FIG. 2B), eight days (FIG. 2C), and comparativemetabolic profiling of hES cells (blue) and conditioned media (yellow)(FIG. 2D).

FIGS. 3A through 3D are photomicrographs of cellular morphology showingthe pluripotent embryonic stem cells following extended culture. Themarker Oct-4 was retained in a similar manner as untreated controls(FIG. 3A=5 days valproate, FIG. 3B=5 days control, FIG. 3C-8 daysvalproate, FIG. 3D-8 days control).

FIGS. 4A through 4B show the results of comparative mass spectrometry inthe presence of chemical standards confirming the chemical identity offolic acid (exact mass 441.14), pyroglutamic acid (exact mass 129.04),glutamate (exact neutral mass 147.05) and kynurenine (exact mass208.08).

FIG. 5 represents the kynurenine metabolism pathway of tryptophan inhumans (Wang et al., 2006, J Biol Chem 281: 22021-22028, publishedelectronically on Jun. 5, 2006).

FIG. 6 illustrates a hierarchical clustering of fold-change differencesfrom 22,573 unique masses and is representative of multiple independentexperiments in which hESCs and neural precursors produced from hESCswere treated with 1 mM valproate. Non-embryonic cells (humanfibroblasts) were used as controls (data not shown). Positive foldchanges are red, negative fold changes are green, and missing data isgrey.

FIG. 7 shows the relative expression of enzymes in the kynurenine andserotonin synthesis pathways in hES cells. INDO, indoleamine 2,3dioxygenase, TDO or TDO2, tryptophan 2,3-dioxygenase. (TDO2 wasupregulated in valproate-treated hES cells in comparison to controls.)AFMID, arylformamidase, TPH1, tryptophan hydroxylase, AADAT,aminoadipate aminotransferase, KYNU, kynunreninase, GAPDH,glyceraldehyde 3-phosphate dehydrogenase, housekeeping control gene.KMO, kynurenine 3-monooxygenase, was not expressed in valproate-treatedcells or controls.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides reagents, including human embryonic stem cells(hESC) or hESC-derived lineage-specific cells, such as neural stemcells, neural precursor cells and neural cells produced therefrom, forassessing developmental toxicity using the human embryonic stem cellmetabolome. Human embryonic stem cells are pluripotent, self-renewingcells isolated directly from preimplantation human embryos thatrecapitulate organogenesis in vitro. Lineage-specific precursor cellsare derived from hES cells and have entered a specific cellular lineage,but yet remain multipotent with regard to cell type within that specificlineage. For example, neural precursors have committed to neuraldifferentiation but yet remain unrestricted as to its neural cell type.Also within the scope of the inventive methods areterminally-differentiated cell types, such as neurons. Biochemicalpathways of human development and disease are active in hESCs and orhESC-derived lineage-specific cells, because they recapitulatedifferentiation into functional somatic cells. Disruption of thesepathways during development contributes to disorders such as neural tubedefects (NTDs) and cognitive impairment. Environmental agents, namelychemicals or drugs, participate in the ontogenesis of certain acquiredcongenital disorders. The question of which pathways during early humandevelopment are particularly susceptible to the effects of theenvironment remains unsolved.

The metabolome, defined as the total dynamic set of cellular metabolitespresent in cells, is a product of health or disease/insult states.Metabolomics is particularly sensitive to environmental effects incomparison to other “omic” areas of study, such as genomics andproteomics. Cellular metabolites include but are not limited to sugars,organic acids, amino acids and fatty acids, particularly those speciessecreted or excreted from cells, that participate in functionalmechanisms of cellular response to pathological or chemical insult.These cellular metabolites serve as biomarkers of disease or toxicresponse and can be detected in biological fluids (Soga et al., 2006, JBiol Chem 281:16768-78; Zhao et al., 2006, Birth Defects Res A Clin MolTeratol 76:230-6), including hESC culture media. Importantly,metabolomic profiling may confirm functional changes that are oftenpredicted by transcriptomics and proteomics.

However, because it was known that hESCs are highly sensitive to theculture microenvironment (Levenstein et al., 2005, Stem Cells 24:568-574; Li et al., 2005, Biotechnol Bioeng 91:688-698.), theirapplication as a source of predictive biomarkers in response to chemicalcompounds, including toxins, teratogens and particularly pharmaceuticalagents, drug lead compounds and candidate compounds in drug development,and their usefulness in establishing in vitro models of disease anddevelopment was uncertain, inter alia because those of skill in the artcould anticipate that exposure to an exogenous chemicals could be highlydetrimental to survival of hES cells and preclude obtaining usefulinformation from them. This concern has turned out not to be justified.

As used herein, the term “human embryonic stem cells (hESCs)” isintended to include undifferentiated stem cells originally derived fromthe inner cell mass of developing blastocysts, and specificallypluripotent, undifferentiated human stem cells andpartially-differentiated cell types thereof (e.g., downstreamprogenitors of differentiating hESC). As provided herein, in vitrocultures of hESC are pluripotent and not immortalized, and can beinduced to produce lineage-specific cells and differentiated cell typesusing methods well-established in the art. In preferred embodiments,hESCs useful in the practice of the methods of this invention arederived from preimplantation blastocysts as described by Thomson et al.,in co-owned U.S. Pat. No. 6,200,806. Multiple hESC cell lines arecurrently available in US and UK stem cell banks.

The terms “stem cell progenitor,” “lineage-specific cell,” “hESC derivedcell” and “differentiated cell” as used herein are intended to encompasslineage-specific cells that are differentiated from hES cells such thatthe cells have committed to a specific lineage of diminished potency. Insome embodiments, these lineage-specific precursor cells remainundifferentiated with regard to final cell type. For example, neuronalstem cells are derived from hESCs and have differentiated enough tocommit to neuronal lineage. However, the neuronal precursor retains‘stemness’ in that it retains the potential to develop into any type ofneuronal cell. Additional cell types include terminally-differentiatedcells derived from hESCs or lineage-specific precursor cells, forexample neural cells.

The term “cellular metabolite” as used herein refers to any smallmolecule secreted and/or excreted by a hESC or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells, produced therefrom. In preferred embodiments,cellular metabolites include but are not limited to sugars, organicacids, amino acids, fatty acids, hormones, vitamins, oligopeptides (lessthan about 100 amino acids in length), as well as ionic fragmentsthereof. Cells may also be lysed in order to measure cellular productspresent within the cell. In particular, said cellular metabolites arefrom about 10 to about 3600 Daltons in molecular weight, moreparticularly about 10 to about 1500 Daltons, and yet more particularlyfrom about 100 to about 1000 Daltons.

hESCs are cultured according to the methods of the invention usingstandard methods of cell culture well-known in the art, including, forexample those methods disclosed in Ludwig et al. (2006,:Feeder-independent culture of human embryonic stem cells, Nat Methods3: 637-46.). In preferred embodiments, hESCs are cultured in the absenceof a feeder cell layer during the practice of the inventive methods;however, hESCs may be cultured on feeder cell layer prior to thepractice of the methods of this invention.

The term “administering” as used herein refers to contacting in vitrocultures of hESCs or hESC-derived lineage-specific cells, such as neuralstem cells, neural precursor cells and neural cells produced therefromwith a toxic, teratogenic, or test chemical compound. In a preferredembodiment the dosage of the compound is administered in an amountequivalent to levels achieved or achievable in vivo, for example, inmaternal circulation.

The phrases “identifying cellular metabolites that are differentiallyproduced” or “detecting alterations in the cells or alternations in cellactivity” as used herein include but are not limited to comparisons oftreated hES cells or hESC-derived lineage-specific cells, such as neuralstem cells, neural precursor cells and neural cells, to untreated(control) cells (i.e., cells cultured in the presence (treated) orabsence (untreated) of a toxic, teratogenic, or test chemical compound).Detection or measurement of variations in cellular metabolites, excretedor secreted therefrom, between treated and untreated cells is includedin this definition. In a preferred embodiment, alterations in cells orcell activity are measured by determining a profile of changes incellular metabolites having a molecular weight of less than 3000Daltons, more particularly between 10 and 1500 Daltons, and even moreparticularly between 100 and 1000 Daltons, in a treated versus untreatedcell as illustrated in FIGS. 1A through 1C.

The term “correlating” as used herein refers to the positive correlationor matching of alterations in cellular metabolites including but notlimited to sugars, organic acids, amino acids, fatty acids, and lowmolecular weight compounds excreted or secreted from hES cells orhESC-derived lineage-specific cells, such as neural stem cells, neuralprecursor cells and neural cells, to an in vivo toxic response. Thescreened cellular metabolites can be involved in a wide range ofbiochemical pathways in the cells and related to a variety of biologicalactivities including, but not limited to inflammation, anti-inflammatoryresponse, vasodilation, neuroprotection, oxidative stress, antioxidantactivity, DNA replication and cell cycle control, methylation, andbiosynthesis of, inter alia, nucleotides, carbohydrates, amino acids andlipids, among others. Alterations in specific subsets of cellularmetabolites can correspond to a particular metabolic or developmentalpathway and thus reveal effects of a test compound on in vivodevelopment.

The term “physical separation method” as used herein refers to anymethod known to those with skill in the art sufficient to produce aprofile of changes and differences in small molecules produced in hESCsor hESC-derived lineage-specific cells, such as neural stem cells,neural precursor cells and neural cells, contacted with a toxic,teratogenic or test chemical compound according to the methods of thisinvention. In a preferred embodiment, physical separation methods permitdetection of cellular metabolites including but not limited to sugars,organic acids, amino acids, fatty acids, hormones, vitamins, andoligopeptides, as well as ionic fragments thereof and low molecularweight compounds (preferably with a molecular weight less than 3000Daltons, more particularly between 10 and 1500 Daltons, and even moreparticularly between 100 and 1000 Daltons). In particular embodiments,this analysis is performed by liquid chromatography/electrosprayionization time of flight mass spectrometry (LC/ESI-TOF-MS), however itwill be understood that cellular metabolites as set forth herein can bedetected using alternative spectrometry methods or other methods knownin the art for analyzing these types of cellular compounds in this sizerange.

Data for statistical analysis were extracted from chromatograms (spectraof mass signals) using the Agilent Mass Hunter software (Product No.G3297AA, Agilent Technologies, Inc., Santa Clara, Calif.); it will beunderstood that alternative statistical analysis methods can be used.Masses were binned together if they were within 10 ppm and eluted withina 2 minutes retention time window. A binned mass was considered to bethe same molecule across different LC/ESI-TOF-MS analyses (referred toherein as an “exact mass,” which will be understood to be ±10 ppm).Binning of the data is required for statistical analysis and comparisonof masses across the entire experiment. If multiple peaks with the samemass at the same retention time within a single sample were detected byMass Hunter, they were averaged to assist data analysis. Masses lackinga natural isotopic distribution or with a signal-to-noise ratio of lessthan 3 were removed from the data prior to analysis. One of skill in theart will appreciate that the results from this assay provide relativevalues that are assessed according to annotated values within 10 ppm toprovide an identity for the molecular weight detected. Thus, a massshift within 10 ppm is considered consistent with determining theidentity of a specific cellular metabolite annotated known in the artdue to differences in ionization source and instrumentation, e.g.between different experiments or using different instruments.

As used herein, a mass was considered to be the same acrossLC/ESI-TOF-MS runs using a simple algorithm that first sorts the data bymass and retention time. After sorting, a compound was considered uniqueif it had a retention time difference of less than or equal to threeminutes and a mass difference less than or equal the weighted formula(0.000011×mass). If a series of measurements fit this definition it wasconsidered to be from the same compound. If either the mass or theretention time varied by more than the limits listed above it wasconsidered to be a different compound and given a new uniquedesignation.

Significance tests were determined by performing ANOVAs on the log base2 transformed abundance values of unique compounds present in treatedand untreated media at each time point. A randomized complete blockdesign was used with the ANOVA model including the effects of treatment,experiments, and a residual term, with the following formula:Log₂(abundance_(tb))=treatment_(t)+experiment_(b)+error_(tb).

Missing data were omitted from the test changing the degrees of freedomrather than assuming the missing data were absent. This assumption wasmade because the extensive filtering performed by the Mass Huntersoftware may miss or filter certain peaks because they are below acertain abundance threshold and not zero. The ANOVA F-test wasconsidered significant if its p-value was less than 0.05. Fold changeswere calculated using the least squared means for a given time andtreatment.

The term “biomarker” as used herein refers to cellular metabolites thatexhibit significant alterations between treated and untreated controls.In preferred embodiments, biomarkers are identified as set forth above,by methods including LC/ESI-TOF-MS. Metabolomic biomarkers areidentified by their unique molecular mass and consistency with which themarker is detected in response to a particular toxic, teratogenic ortest chemical compound; thus the actual identity of the underlyingcompound that corresponds to the biomarker is not required for thepractice of this invention. Alternatively, certain biomarkers can beidentified by, for example, gene expression analysis, includingreal-time PCR, RT-PCR, Northern analysis, and in situ hybridization, butthese will not generally fall within the definition of the term“cellular metabolites” as set forth herein.

The basal metabolome of undifferentiated hESCs served as a collection ofbiochemical signatures of functional pathways that are relevant forstemness and self-renewal. Metabolite profiling was conducted onexcreted or secreted cellular metabolites as opposed to intracellularcompounds. Ultimately, biomarkers discovered in vitro are expected to beuseful for analyzing in vivo biofluids such as serum, amniotic fluid andurine, complex mixtures of extracellular biomolecules. This isadvantageous over invasive procedures such as tissue biopsies becausesmall molecules in biofluids can be detected non-invasively (in contrastto intracellular compounds). In addition, processing cellularsupernatant for mass spectrometry is more robust and less laborious thancellular extracts. However, cellular extracts (from, for example, lysedcells) can be utilized in the methods of the invention.

The term “biomarker profile” as used herein refers to a plurality ofbiomarkers identified by the inventive methods. Biomarker profilesaccording to the invention can provide a molecular “fingerprint of thetoxic and teratogenic effects of a test compound and convey whatcellular metabolites, specifically excreted and secreted cellularmetabolites, were significantly altered following test compoundadministration to hESCs or hESC-derived lineage-specific cells, such asneural stem cells, neural precursor cells and neural cells. In theseembodiments, each of the plurality of biomarkers is characterized andidentified by its unique molecular mass and consistency with which thebiomarker is detected in response to a particular toxic, teratogenic ortest chemical compound; thus the actual identity of the underlyingcompound that corresponds to the biomarker is not required for thepractice of this invention.

The term “biomarker portfolio” as used herein refers to a collection ofindividual biomarker profiles. The biomarker portfolios may be used asreferences to compare biomarker profiles from novel or unknowncompounds. Biomarker portfolios can be used for identifying commonpathways, particularly metabolic or developmental pathways, of toxic orteratogenic response.

These results set forth herein demonstrated that human embryonic stemcell metabolomics, and metabolomics from hESC-derived lineage-specificcells, such as neural stem cells, neural precursor cells and neuralcells, can be used in biomarker discovery and pathway identification.Metabolomics detected small molecules secreted by hESCs or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells, produced therefrom and the identified biomarkerscan be used for at least two purposes: first, to determine specificmetabolic or developmental pathways that respond to or are affected bytoxin or teratogen exposure, particularly said pathways utilized oraffected during early development that are sensitive to toxic,teratogenic or test chemical compounds that are developmental disruptorsand participate in the ontogenesis of birth defects; and second, toprovide cellular metabolites that can be measured in biofluids to assistmanagement and diagnosis of toxic exposure, birth defects or otherdisease.

A biomarker portfolio from hESCs or hESC-derived lineage-specific cells,such as neural stem cells, neural precursor cells and neural cells,produced therefrom can also serve as a high throughput screening tool inpreclinical phases of drug discovery. In addition, this approach can beused to detect detrimental effects of environmental (heavy metals,industrial waste products) and nutritional chemicals (such as alcohol)on human development. Ultimately, the methods of this inventionutilizing the hESC metabolome or the metabolome of or hESC-derivedlineage-specific cells, such as neural stem cells, neural precursorcells and neural cells, can assist pharmaceutical, biotechnology andenvironmental agencies on decision-making towards development ofcompounds and critical doses for human exposure. The integration ofchemical biology to embryonic stem cell technology also offers uniqueopportunities to strengthen understanding of human development anddisease. Metabolomics of cells differentiated from hESC should servesimilar roles and be useful for elucidating mechanisms of toxicity anddisease with greater sensitivity for particular cell or tissue types,and in a human-specific manner. For example, key metabolic pathways,including as set forth herein folate, glutamate and tryptophan synthesisand degradation, may be differentially disrupted in earlier versus laterstages of human development. In addition, metabolite profiles of neuralprecursor cells or neuronal cell populations can reveal biomarkers ofneurodevelopmental disorders in target cell types. The association ofmetabolomics to stem cell biology can inform the mechanisms of action offolic acid and neural tube defects in the early human embryo.

Biomarker portfolios produced using the hESC-dependent and hESC-derivedlineage-specific cell-dependent methods of this invention can also beused in high throughput screening methods for preclinical assessment ofdrug candidates and lead compounds in drug discovery. This aspect of theinventive methods produces minimal impact on industry resources incomparison to current developmental toxicology models, sinceimplementation of this technology does not require experimental animals.The resulting positive impact on productivity enables research teams inthe pharmaceutical industry to select and advance compounds intoexploratory development with greater confidence and decreased risk ofencountering adverse developmental effects.

The term “developmental pathway” as used herein refers to developmentalor metabolic pathways in embryonic and fetal development.

“Supernatant” as used herein may include but is not limited toextracellular media, co-cultured media, cells, or a solution offractionated or lysed cells.

Cellular metabolite profiles obtained from analysis of toxins,teratogens, alcohol, and test chemical compounds can be used to composea library of biomarker portfolios. These portfolios can then be used asa reference for toxicological analysis of unknown chemical compounds. Asimilar strategy has been validated as a means to determine cellularchanges that arise in response to chemicals in non-hESC systems (Daston& Nacliff, 2005, Reprod Toxicology 19:381-94; Fella et al., 2005,Proteomics 5:1914-21). Metabolic profiles of novel compounds can becompared to known biomarker portfolios to identify common mechanisms oftoxic response. This approach can reveal functional markers of toxicresponse, which serve as screening molecules that are shared at least inpart as a consequence of exposure to various different toxic andteratogenic compounds. Such hESC-derived small molecules can be used asmeasurable mediators of toxic response that refine or replace costly andcomplex screening systems (such as in vivo animal models) and have theadditional advantage of being specific for human cells and humanmetabolic and developmental pathways.

EXAMPLES

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. They are set forth forexplanatory purposes only, and are not to be taken as limiting theinvention.

Example 1 Developmental Toxicology Screening

To demonstrate the efficacy of hESCs as a model system for developmentaltoxicity testing, hESCs were treated with a known teratogen, valproate(VPA). Valproate is a common mood stabilizer and anti-convulsant drugwith clinical indications in epilepsy and bipolar disorder (Williams etal., 2001, Dev Med Child Neuro 43:202-6) that has been associated withdevelopmental abnormalities (Meador et al., 2006, Neurology 67:407-412). The mechanism by which valproate produces developmentaldefects, however, is not fully understood, despite the increasedsusceptibility of the nervous system (Bjerkedal et al., 1982, Lance2:109: Wyszynski et al., 2005, Neurology 64:961-5; Rasalam et al., 2005,Dev Med Child Neuro 47:551-555). Exposure to valproate results in apronounced increase in spina bifida and neural tube defects (NTDs;Bjerkedal et al., 1982, Lancet 2:109) at ten-to-twenty times that of thegeneral population, as well as cognitive disorders such as autism (Adabet al., 2004, J Neurol Neurosurg Psychiatry 75:1575-83). However, sinceVPA is an anti-convulsant drug with clinical indications in epilepsy andbipolar disorder (Williams et al., 2001, Dev Med Child Neurol43:202-06), treatment generally must be sustained throughout pregnancy.

Folic acid supplementation prior to pregnancy reduces the incidence ofspina bifida by 70% (Shaw et al., 1995, Epidemiology 6:219-226) althoughits precise mechanism of action is unknown. In addition, homocysteineand glutathione have also been implicated in NTDs (Zhao et al., 2006,Birth Defects Res A Clin Mol Teratol 76:230-6). Thus, metaboliteprofiles of folate and related pathways were candidates for changes inresponse to valproate. In the results set forth herein, folic acid wassignificantly increased (by 16%) in the extracellular media of hES cellstreated with valproate (p=0.022 at eight days, Table 3 and FIG. 4) butnot its derivative dihydrofolate. Since mammalian cells do notsynthesize folic acid, valproate may act by interfering with cellularuptake of folic acid.

Exposure of hESCs was performed as follows. H1 hESC (passage 41) werecultured on Matrigel (BD Scientific, San Jose, Calif.) in the absence ofa feeder layer. hESCs were maintained in conditioned medium (CM)collected from mouse embryonic fibroblasts (MEFs) (80% DMEM/F12,Invitrogen, Carlsbad, Calif.) and 20% KNOCKOUT serum replacement(Invitrogen) supplemented with 1 mM L-glutamine (Invitrogen), 1% MEMnon-essential amino acids (Invitrogen), and 0.1 mM 2-mercaptoethanol(Sigma, Chemical Co., St. Louis, Mo.). Prior to feeding hESCs, theculture medium was supplemented with 4 ng/mL human recombinant basicfibroblast growth factor (Invitrogen). hESCs were passaged when thewells were ˜80% confluent. To passage, hESCs were incubated in a 1 mg/mLdispase (Invitrogen)/DMEM/F12 solution for 7-10 minutes at 37° C. Afterthis treatment hESCs were washed and seeded on fresh Matrigel coatedplates. In parallel studies disclosed herein, H1 and H9 cells werecultured in defined medium known as TeSR (Ludwig et al., 2006, Id.).

H1 and H9 (equivalent to NIH code WA01/WA09) hESC were treated withvalproate (VPA) (22 μM and 1 mM) (Sigma # P4543) according to theprocedure outlined below; each experiment involved three separate VPAtreatments, and each treatment group had a parallel control group with atotal of six 6-well culture dishes (Nunc, Naperville, Ill.) perexperiment (two 6-well culture dishes per treatment). Treatment 1(labeled 24H) exposed hESC cells to 1 mM VPA (Sigma) for 24 hoursfollowed by collection of supernatant and cell pellets. In a secondtreatment group (labeled 4D), hESC cells were exposed to 22 μM or 1 mMVPA for 4 days and harvested on day 4. In a third treatment group(labeled extended culture, EC), hESC cells received 22 μM or 1 mM VPAfor 4 days followed by culture in standard hESC media for an additionalfour days. For this group, cells and supernatant were harvested on dayeight.

To assess the effects of teratogenic VPA treatment on hESCs, the treatedcells were analyzed as set forth below to determine changes in a totaldynamic set of small molecules present in cells according to health anddisease or insult states. Small molecules including but not limited tosugars, organic acids, amino acids, fatty acids, hormones, vitamins,oligopeptides (less than about 100 amino acids in length), as well asionic fragments thereof and signaling low molecular weight compoundswere known to participate in and reveal functional mechanisms ofcellular response to pathological or chemical insult. These analyseswere also used to identify active pathways following molecular changespredicted by other analyses including for example transcriptomics andproteomics.

Supernatant from VPA-treated and control hESCs were subjected to liquidchromatography and electrospray ionization time of flight massspectrometry (LC/ESI-TOF-MS) to assess changes and differences in smallmolecules (as defined herein) produced by the cells in the presence andabsence of VPA treatment. Supernatant was collected from control andtreated plates of hESCs at 24H, 4D, and 8D, and CM was collected as a“no treatment” control. The supernatant and media were stored at −80° C.until preparation for mass spectrometry analysis. For analysis, sampleswere prepared in a 20% Acetonitrile (Fisher Scientific Co., Pittsburgh,Pa.) solution (comprising 500 μL of supernatant, 400 μL acetonitrile and1.1 mL distilled water) and centrifuged through a Millipore 3 kDaCentricon column (Millipore, Billerica, Mass.) for 3 hours at 4575×g toremove proteins. The flow-through was retained for analysis, as itcontains small molecules free of high molecular weight compounds such asproteins. In each analysis, three replicates for each sample wereinjected into a 2.1×200 mm C18 column using a 90 minute gradient from 5%Acetonitrile, 95% Water, 0.1% Formic Acid to 100% Acetonitrile, 0.1%Formic Acid at a flow rate of 40 μL/min. ESI-TOF-MS (TOF) was performedon the flow-through using an Agilent ESI-TOF mass spectrometer. Data wascollected from 100-3600 m/z, and particularly in the 0-1500 m/z range.The raw data was analyzed to identify the separated small moleculesusing a computer compilation and analysis program (Mass Hunter) providedby the manufacturer and according to manufacturer's instructions(Agilent; statistical analyses were performed as described above in theDetailed Description and Preferred Embodiments. This analysis generatedlists of retention time/accurate mass pair feature. Another program(Mass Profiler, Agilent) was used to compare multiple run sets to findion intensity changes of features that changed between the sampleconditions. Significance tests were determined by performing ANOVAs onthe log base 2 transformed abundance values of unique compounds presentin treated and untreated media at each time point.

The plurality of small molecules identified using these methods werethen compared with exact mass and retention time from ESI-TOF-MS usingpublic databases (for example, athttp://metlin.scripps.edu.,www.nist.gov/srd/chemistry.htm;http://www.metabolomics.ca/). Mass spectrometry analysis also includedpredicted chemical structures of small molecules based upon exact mass,although currently-available public databases do not in every instanceinclude matching small molecules due to database limitations. Inaddition, more comprehensive private databases are available forcomparative analysis, such as the NIST/EPA/NIH Mass Spectral Library:05. NIST ASCII Version.

The results of these analyses are shown in FIGS. 1A through 1C and FIG.2A through 2C. In FIGS. 1A through 1C each feature on the plotcorresponds to a small molecule with specific exact mass and retentiontime. The plots summarize significant differences found between treated(blue) and untreated (red) groups at different time points. As shown inthe Figure, at 24 hours (24H) there was consistent down-regulation ofthe secreted biomolecules in treated (blue) cells in comparison tountreated (red) controls. At four days (4D) and eight days (EC), treated(blue) cells secreted a higher number of small molecules in comparisonto untreated cells (red); said small molecules were thus considered ascandidate biomarkers. In particular, metabolites from the folatepathway, including tetrahydrofolate (exact mass 444) and dihydrofolate(exact mass 441) were detected. These findings were consideredsignificant, since they show for the first time that hESCs contactedwith a known teratogen (VPA) that causes a birth defect (spina bifida)respond by up-regulating a metabolic pathway that produces a compound(folate) known to ameliorate the effects of the teratogen whenadministered to a woman bearing a developing embryo or fetus.

Further, the results shown in FIGS. 1A through 1C revealed approximately40 small molecules that were absent in treated groups, suggesting thatmultiple cellular pathways were “silenced” in response to VPA at 24hours in comparison to untreated controls. At four and eight days aftertreatment, however, multiple candidate biomarkers were upregulated intreated versus untreated human embryonic stem cells; these results areshown in Table 1. Candidate biomarkers were identified as smallmolecules showing a change in treated versus untreated cells measured tobe at least a two-fold difference. In many instances, these smallmolecules are absent or detected at very low concentrations in untreatedhuman embryonic stem cells.

These studies demonstrated that the claimed methods for assessingdevelopmental toxicity and the identification of biomarkers using hESCsprovided robust information on changes in small molecule content ofcells in response to being contacted with a known teratogen, VPA. Theresults concerning a compound (VPA) that is involved in the etiology ofspina bifida and neural tube defects (NTDs)(Bjerkedal et al., 1982,Lancet 2:109) when exposed to a developing human conceptus areparticularly striking. The results shown here indicated a markedincrease (2 to 8 fold) in key metabolites of the folate pathway(dihydrofolic acid, tetrahydrofolic acid, S-adenosylmethionine)following treatment with VPA (in comparison to untreated cells). Thesemethods were reproducible, having been repeated with consistent resultsobtained in three independent studies using hESCs and on non-embryoniccells (human fibroblasts) as controls (data not shown), and suggested aheretofore unknown adaptive response of the fetus to thechemical/environmental insult and identified sensitive markers for saidinsult(s).

The mechanism for VPA developmental defects, however, is not fullyunderstood despite the fact that the nervous system is particularlysensitive to its effects (Bjerkedal et al., 1982, Lancet 2:109; Naritaet al., 2000, Pediatric Res 52:576-79; Rasalam et al., 2005, Dev MedChild Neurol 47:551-55). Folic acid supplementation prior to pregnancyprevents the incidence of spina bifida by 70% (Shaw et al., 1995,Epidemiology 6:219-226), although the exact mechanism of action is alsounknown. The information obtained herein can be used to elucidatemechanisms of action of folic acid and neural tube defects in the earlyhuman embryo. These methods can also be applied to other knownteratogens, such as retinoic acid, warfarin, and thalidomide (Franks etal., 2004, Lancet 363:1802-11) to validate the predictive ability ofhESCs using the methods of the invention.

TABLE 1 Candidate small molecules (biomarkers) of developmental toxicitydetected in undifferentiated human embryonic stem cells treated with 1mM valproate in comparison to untreated controls. Change in VPA TreatedhESCs in comparison to untreated controls Exact 24 4 8 mass RT HoursDays Days Candidate Biomarker 355.066 16 UP SAM S-ADENOSYLME-THIONINAMINE 355.12 30 UP SAM 381.1574 12 UP UP GLUTATHIONE 398.21 39DOWN UP SAM OXOBUTANOATE 441.8831 12 UP DIHYDROFOLIC ACID 444.1729 17 UPUP TETRAHYDROFOLIC ACID 472.16 17 ZERO UP UP TETRAHYDROFOLATE 612.15 17DOWN DOWN UP GLUTATHIONE OXIDIZED RT = retention time Small moleculedetection was conducted with LC/ESI-TOF-MS in triplicate samples ofsupernatant processed independently.

As discussed above, metabolite profiles were determined at 24 hours,four days and eight days after valproate treatment. At four days aftertreatment, multiple candidate biomarkers were upregulated in treatedversus untreated human embryonic stem cells (shown in FIGS. 2A through2C). In addition to the results set forth above regarding increasedlevels of certain metabolites, multiple metabolite peaks weredown-regulated in response to valproate at 24 hours in comparison tountreated controls (FIGS. 2A through 2C).

hESCs were cultured in conditioned media from mouse embryonicfibroblasts, which generated 1277 of the 3241 measured compounds. Manymetabolites in human development and disease are likely present inconditioned media from mouse embryonic fibroblasts due to commonmetabolic pathways. Rigorous investigation is required to validatecandidate biomarkers that are not exclusive to hESCs and are alsopresent in the media.

Example 2 Gene Expression Analysis

The efficacy of the analysis shown in Example 1 was confirmed by geneexpression studies, wherein changes in gene expression were observedfollowing VPA treatment of hESCs. VPA treatment was not detrimental tohESCs, which remained viable for multiple passages following teratogenexposure, thus enabling gene expression analysis to be performed.

Treated and control H1/NIH code WA01 hES cells (passage 41) wereanalyzed by real-time PCR, and each treatment group was paired with acorresponding control group that received the standard growth mediacombination of CM+bFGF without VPA. In these studies, total cellular RNAwas extracted from cells harvested at 24 hours (24H), 4 days (4D) and 8days (EC) using the RNA Easy Kit (Qiagen, Valencia, Calif.) according tothe manufacturer's instructions.

Expression levels of candidate test genes and a housekeeping gene(Beta-2-microglobulin) were evaluated by quantitative real-time PCRusing a DNA Engine-Opticon 2 Detection System (MJ Research, Watertown,Mass.). The housekeeping gene acts as an internal control fornormalization of RNA levels. The primers used for real-time PCRreactions were designed using Beacon Designer software (Premier BiosoftInternational, Palo Alto, Calif.). RNA was reverse transcribed usingiScript cDNA Synthesis kit (Bio-Rad, Hercules, Calif.), wherein eachcDNA synthesis reaction (20 μL) included 4 μL of 5× iScript reactionmix, 1 μL of iScript reverse transcriptase, and 2 μL of RNA. PCR wasperformed on cDNA in PCR reaction mixtures (25 μL) each containing 12.5μL of Supermix (contains dNTPs, Taq DNA polymerase, SYBR Green I, andfluorescein), 250 nM forward primer, 250 nM reverse primer, and 1.6 μLRT-PCR products. Melting curve analysis and agarose gel electrophoresiswere performed after real-time PCR reaction to monitor PCR specificity,wherein PCR products were detected with SYBR Green I using the iQ SYBRGreen Supermix kit (Bio-Rad).

Quantifying the relative expression of real-time PCR was performed usingthe 2-AACt method (Livak & Schmittgen, 2001, Methods 25:402-8), and ageneral linear model was employed to fit the expression data. The PROCGLM procedure in SAS (version 8.2; SAS Institute, Cary, N.C.) was usedto estimate least squares means in expression between treated anduntreated hESCs and P<0.05 was considered statistically significant

Real-time PCR was conducted on samples from 24 hours (24H), 4 days (4D)and 8 days (EC) after VPA treatment to investigate expression levels ofepigenetic regulators (such as DNA methyltransferase-1, DNMT-1, BMI-1,EED) and critical transcription factors responsible for embryonicpatterning and neurodevelopment (RUNX2, BMP7, FGF8, CBX2, GLI3, SSH andSP8) in human embryonic stem cells. These experiments showed hESCstreated with VPA were subject to marked changes in their transcriptionalactivity following teratogen treatment. VPA induced overall marked (2 to30 fold) downregulation of transcription levels as early as 24 hoursafter exposure in all genes tested (with the exception of DNMT-1 andShh). At 4 days after treatment, however, expression of the ubiquitousDNA methyltransferase-1 was almost abolished, and sonic hedgehog, whichis absolutely critical for neurogenesis (Ye et al., 1998, Cell93:755-66), was down-regulated five-fold in comparison to untreatedcontrols. At 8 days after VPA treatment, the majority of the genes wereupregulated in comparison to untreated controls.

These results embodied two major implications for developmentaltoxicology. First, VPA induced persistent changes in key epigeneticmodulators that also participate in differentiation of other tissues,such as DNMT-1 and the polycomb family member EED. Second, the effectsof teratogens persisted in hESCs during critical stages of neurogenesisand organogenesis. For example, genes whose expression was affected asshown herein (including sonic hedgehog and FGF-8) are known to be masterregulators of differentiation of serotonergic neurons in the brain (Yeet al., 1998, Cell 93:755-66). Of particular notice is the fact thatDNMT-1 expression is almost abolished at four days after treatment. Invivo, disruption of this enzyme is lethal to embryos, since it is themajor maintenance methyltransferase during DNA replication (Li et al.,1992, Cell 69:915-26).

Following teratogen exposure, temporal-specific alterations indevelopmental gene expression were observed. Developmental genes differin their susceptibility to teratogens at different times. Thisindication may be critical to understanding specificity of epigeneticdisruptors on certain organs or tissues. RUNX2, for example, is atranscriptional activator of bone development (Napierala et al., 2005,Mol Genet Metab 86:257-68), and is more sensitive to VPA-mediatedup-regulation at very early or late stages following exposure. Real-timePCR results from hESCs disclosed herein were correlated to previousfindings in vivo in mice (Okada et al., 2004, Birth Defects Res A ClinMol Teratol 70:870-879) and rats (Miyazaki et al., 2005, Int J DevlNeuroscience 23:287-97). In these animal studies, VPA inhibited theexpression of Polycomb genes, Eed, Bmil and Cbx2 and induceddownregulation of Shh while FGF8 levels remained unchanged. The resultsshown here at four days following VPA treatment (Table 2) wereconsistent with these observations using other developmental modelsystems.

TABLE 2 1 mM VPA treatment resulted in marked changes in the expressionof epigenetic regulators and developmental genes that are critical forembryonic patterning and differentiation of neurons. 2-24 H 4-4 D 1-24 HVPA 3-4 D VPA 5-EC 6-EC VPA Gene control treated control treated controltreated BMI-1 0.543 0.252 1.651 0.112 1.020 1.071 DNMT1 0.664 0.6241.742 0.002 0.731 1.124 EED 0.757 0.342 1.501 0.185 1.381 1.769 H190.207 0.006 0.144 0.846 10.660 2.756 RUNX2 0.325 1.769 5.198 4.020 1.1772.434 BMP7 0.511 0.093 0.397 0.342 0.731 1.664 FGF8 2.544 0.801 0.3840.314 0.16 0.837 CBX2 1.113 0.245 1.221 1.1881 0.81 1.946 GLI3 0.2020.015 0.016 0.774 0.950 1.918 Shh 0.562 0.562 2.772 0.533 1.369 — SP80.49 0.235 0.25 1.703 2.132 5.808 Gene expression levels are relative toa housekeeping gene (target gene/Beta-2-Microglobulin).

Example 3 Human Embryonic Stem Cell Metabolome: Metabolite ProfilesFollowing Teratogen Exposure

Exposure of hES cells to the teratogen valproate induced significantchanges in different metabolic pathways, including pathways importantduring pregnancy and development. An alternative metabolic pathwayactivated during pregnancy are shown in FIG. 5, wherein tryptophan isconverted to kynurenine. To investigate this aspect of the invention,hESCs were cultured as described in Example 1, and the procedure forvalproate treatment was performed as described therein. Treatment 1 (24hours) exposed hES cells to 22 μM valproate for 24 hours followed bycollection of supernatant and cell pellets. In the second treatmentgroup (4 days), hES cells were exposed to 22 μM valproate for 4 days andharvested on day 4. In the third treatment or extended culture (EC, 8days), hES cells received valproate for 4 days followed by culture instandard hES cell media for an additional four days. Cells andsupernatant were harvested on day eight. Each treatment had a parallelcontrol group with a total of six 6-well culture dishes per experiment(two 6-well culture dishes per treatment).

Metabolome analysis was performed as described in Example 1 (Wu andMcAllister, 2003, J Mass Spectrom 38:1043-53). Complex mixtures wereseparated by liquid chromatography (LC) prior to electrospray ionization(ESI) time of flight (TOF) mass spectrometry according to the methodsdescribed in this Example and Example 1. Mass Hunter (Agilent) softwarewas applied to deconvolute the data and determine the abundance of eachmass. Data were extracted from the entire mass spectrum using the m/zrange of 0 to 1500 and the top 2 million most abundant mass peaks fromeach sample were used for data deconvolution. The minimumsignal-to-noise ratio was set to 5. The masses with a minimum relativeabundance greater than 0.1% were exported from the Mass Hunter softwareand used for further analysis.

hESCs treated with 22 μM valproate resulted in 3,241 detected masssignals 42 injections. Of the total of 3,241 mass signals detected inthese experiments, 1,963 compounds were measured solely in hES cells and1,278 compounds were also present in conditioned media; 443 of thesewere only measured in 1 of 42 injections. 110 compounds (3%) hadstatistically-significant differences in at least one time point invalproate-treated hESCs compared with control. Fold changes as high asseven- to thirteen-fold were measured after valproate treatment, butthese mass signals exhibited high variability across experiments.Representative masses identified following treatment of cells with 1 mMand 22 μM VPA are summarized in Tables 3 and 4, respectively. Severalpeaks (1,963) were detected in hES cells but not in conditioned media.One of these small molecules was kynurenine, a compound produced by analternative tryptophan metabolic pathway, activated during pregnancy andimmune response. The levels of kynurenine increased by 44% (pvalue=0.004 at four days, Table 5) following valproate treatment.Kynurenine was detected exclusively in hES cells and absent inconditioned media. The chemical identity of this peak was confirmed bycomparative mass spectrometry in the presence of the chemical standard(FIG. 4).

The results of these experiments suggested that kynurenine is acandidate biomarker for neurodevelopmental disorders, in particularthose originated by exposure of the human embryo to anti-epileptic drugssuch as VPA (Ornoy et al., 2006, Reproductive Toxicol 21:399-409).Strikingly, recent studies have suggested that kynurenine metabolism maybe a novel target for the mechanism of action of anti-epileptic drugs(Kocki et al., 2006, Eur J Pharmacol 542:147-51). Cognitive andbehavioral disorders are known adverse effects of antiepileptic exposureduring pregnancy. Tryptophan is the precursor of serotonin, a keyneurotransmitter in the pathogenesis of these and other diseases, suchas depression. In addition, increased plasma levels of kynurenine havebeen linked to postpartum depression (Kohl et al., 2005, J Affect Disord86:135-42). The alteration in tryptophan metabolism detected herein is ameans for examining novel mechanisms in pathogenesis ofserotonin-related behavioral disorders such as autism (Chugani, 2004,Ment Retard Dev Disabil Res Rev 10:112-116). An increase in kynureninelevels during development may reduce the bioavailability of tryptophanand consequently serotonin, leading to cognitive dysfunction.

Glutamate and pyroglutamic acid were also elevated in hESCs treated withvalproate. Glutamate and pyroglutamic acid were elevated in response tovalproate (20% and 27%, respectively), although only pyroglutamic acidexhibited statistically significant changes (p=0.021 at 4 days, FIGS. 3Athrough 3D). Glutathione (GSH) is metabolized bygamma-glutamyltranspeptidase into glutamate, a neurotransmitter of NMDAreceptors, and cysteinylglycine (Cys-Gly). Glutathione (exact neutralmass 612.15) and S-adenosyl-homocysteine (exact neutral mass 384.12)were detected at very low levels in comparison to other mass signals(data not shown). For these experiments for low level detection, smallmolecules were identified by comparative ESI-TOF-MS with chemicalstandards that were “spiked” into conditioned media at differentconcentrations and used to confirm neutral exact masses and retentiontimes of experimental mass signals (FIGS. 3A through 3D). Neutral exactmasses and/or empirical chemical formulas generated by ESI-TOF-MS weresearched in public databases (including for example,metlin.scripps.edu., www.nist.gov/srd/chemistry.htm, www.metabolmics.ca)for candidate compounds.

These results suggested that valproate affects the glutamate synthesispathway in the developing human embryo. The affinity of anti-epilepticdrugs towards glutamate targets has been previously suggested (Rogawskiand Loscher, 2004, Nat Rev Neurosci 2004 5:553-64). Abnormal levels ofglutamate metabolites were measured in maternal serum and amniotic fluidof pregnant women whose infants were diagnosed spina bifida (Groenen etal., 2004, Eur J Obstet Gynecol Reprod Biol.; 112:16-23) with nuclearmagnetic resonance (NMR). The levels of glutamine and hydroxyprolinewere significantly higher in NTDs, and as a result the hESC methodsprovided herein provide a robust resource to model in vivo alterationsof development.

TABLE 5 Changes in metabolic profiles of four compounds in hES cellstreated with valproate versus untreated controls at 24 hours (24 h), 4days (4 D), and eight days (8 D) after treatment. 24 h P- 4 D P- 8 D P-Molecule value 24 h fold value 4 D fold value 8 D fold Mass RTPyroglutamic 0.242 57% 0.021 27% increase 0.917 3% 129.0426 19.9 aciddecrease decrease Folic acid 0.638 3% 0.626  4% increase 0.022 16%441.1395 32.7 increase increase Glutamate 0.969 1% 0.108 24% increase0.651 10% 147.0535 20.0 increase increase Kynurenine 0.087 29% 0.004 44%increase N.D. N.D. 208.0850 25.9 increase RT = retention time Foldchanges are represented as percent difference of the least squared meansof valproate treated and untreated hES cells. p-values were determinedby ANOVA. The mass is the average neutral mass detected by ESI-TOF-MSand the RT is the average retention time the molecule eluted at.P-values less than 0.05 are in bold.

Example 4 Kynurenine: Biomarker for Diagnosis and Treatment ofDevelopmental Toxicity and CNS Disorders

Kynurenine was shown in Example 3 to be detected in valproate-treatedhES cells. Kynurenine (along with glutamate and pyroglutamic acid) wasdifferentially produced in valproate-treated human embryonic stem cells(hES) versus controls. Kynurenine is a novel biomarker useful for theidentification of neurodevelopmental disorders in infants and in vitrodevelopmental toxicity of chemicals. This example describes theidentification of biomarkers for neurodevelopmental disorders, includingcellular products differentially produced in teratogen-treated hESCs.

The amino acid tryptophan (TRP) is a precursor of the neurotransmitterserotonin, a key mediator of numerous CNS disorders, such as depression,neurodegeneration and cognitive impairment. Tryptophan catabolism intokynurenic acid is an alternative route for tryptophan metabolism (FIG.5), that is activated in specific circumstances such as inflammatoryresponse or pregnancy. Up-regulation of the kynurenine pathway iscorrelated with psychosis in adult diseases such as schizophrenia andbipolar disorder, an indication that increased levels of pathwayintermediates may trigger psychotic features (Miller et al., 2006, BrainRes 16:25-37). Significantly, metabolism using the kynurenine pathway isaccompanied by decreased tryptophan metabolism using the serotoninpathway (in the absence of exogenous tryptophan, an essential amino acidnot synthesized by mammals including man). An increase in kynureninelevels during development can reduce the bioavailability of tryptophanand consequently serotonin, leading to cognitive dysfunction.

In addition, kynurenic acid (KYNA), one of the end products of thistryptophan metabolic pathway, is an antagonist of glutamateneurotransmission and N-methyl-D-aspartate (NMDA) receptors. Recentstudies have demonstrated that kynurenic acid is a druggable target viaits role in the activation of the previously orphan GPCR receptor GPR35(Wang et al., 2006, J Biol Chem 281:22021-8). Quinolinic acid (QUIN),another end product of the pathway (FIG. 5), and 3-hydroxy-kynurenine,an intermediate, act as neurotoxicants (Guillemin et al., 2005, JNeuroinflammation 26:16; Chiarugui et al., 2001, J Neurochem 77:1310-8).QUIN is involved in the pathogenesis of Alzheimer's disease where itsneurotoxicity may be involved in increased inflammation and inconvulsions by interacting with the N-methyl-D-aspartate (NMDA) receptorcomplex, a type of glutamate receptor (Guillemin et al., 2002, JNeuroinflammation 26:16,;Nemeth et al., 2005, Curr Neurovasc Res2:249-60). Kynurenin (KYN), another pathway intermediate, is synthesizedin the brain and is transported across the blood-brain barrier (Nemethet al., 2005, Curr Neurovasc Res 2:249-60). KYN is metabolized to theneurotoxic quinolinic acid (QUIN) and the neuroprotective kynurenic acid(KYNA) (FIG. 5). Increased serum levels of KYN have been correlated toclinical manifestation of depression with different etiologies, such aspost-partum disorder (Kohl et al., 2005, J Affect Disord 86:135-42) andinterferon-alpha treatment (Capuron et al., 2003, Biol Psychiatry54:906-14).

Exposure of hES cells to valproate, a disruptor of human development,induced significant changes in different metabolic pathways, includingthe production of kynurenine (exact neutral mass 208.08), which wassignificantly upregulated in response to valproate as detected by liquidchromatography electrospray ionization time of flight mass spectrometry(LC/ESI-TOF-MS) as described in Example 4. Additionally, novel chemicalentities, having exact neutral masses of 328.058, 336.163, 343.080, weredetected and are not yet catalogued in public databases.

When neural precursors derived from hESCs were exposed to 1 mMvalproate, a marked decrease in both serotonin (176.0946) andindoleacetaldehyde (159.0689), a downstream sub-product of serotoningenerated by monoaminoxoidase activity (MAO) was observed (Table 6).Glutamate and pyroglutamic acid or hydroxyproline (p=0.021) were alsoelevated in hES cells treated with valproate. These results suggest thatvalproate affects the glutamate synthesis pathway in the developinghuman embryo. This finding emulates in vivo neurophysiology, wherecompounds from the kynurenine pathway modulate activity at NMDAglutamate receptors and produce epileptic phenotypes, including seizures(Perkins and Stone, 1982, Brain Res 247:184-187.).

As a consequence of the identification of kynurenine herein, chemicalinhibitors of kynurenine synthesis can be used as novel therapeutics inmood disorders; for example, small molecules that antagonize indoleamine2,3-dioxygenase (IDO) or kynurenine formylase activities, which convertstryptophan (TRP) into kynurenine (KYN) Inhibition of TRP catabolism toKYN can be used to ameliorate disease symptoms in cognitive andneurodegenerative disorders by increasing serotonin levels, via elevatedsynthesis of this neurotransmitter or reduced depletion through thekynurenine pathway.

Collectively, the metabolite changes detected in hES cells in responseto valproate converge functionally towards folate, kynurenine andglutamate pathways. FIG. 6 illustrates the hierarchical clustering ofthe fold change differences from 22,573 unique masses. Changes in theabove-mentioned pathways were consistent and reproducible in multipleindependent studies of 1 mM VPA treated hESCs, and neural precursorsproduced from hESCs (FIG. 6).

Example 5 Gene Expression Analysis of Kynurenine Pathway

The efficacy of the analysis in Example 4 was confirmed by geneexpression studies, wherein changes in gene expression were observedfollowing VPA treatment of hESC. Valproate treatment of human embryonicstem cells induced a marked upregulation in the small moleculekynurenine, an intermediate metabolite in the catabolism of tryptophan.Tryptophan is the precursor of the neurotransmitter serotonin (5HT).Thus, whether expression of enzymes in the metabolism of tryptophan tokynurenine and its opposite route, serotonin synthesis, was altered inhuman embryonic stem cells was investigated to examine the mechanisticproperties of the kynurenine pathway and its response to valproate.

Human embryonic stem cells treated with 1 mM valproate and untreatedcontrols were harvested at four days after treatment and stored at −80°C. prior to RNA isolation using RNeasy (Qiagen). 5 μg of RNA templateswere reverse transcribed and amplified (QIAGEN OneStep RT-PCR) accordingto the manufacturer's instructions using primers designed fortranscribed human sequences of the following genes: INDO, indoleamine2,3 dioxygenase, TDO or TDO2, tryptophan 2,3-dioxygenase, AFMID,arylformamidase, TPH1, tryptophan hydroxylase the rate-limiting enzymein serotonin biosynthesis, AADAT, aminoadipate aminotransferase, KYNU,kynunreninase, KMO, kynurenine 3-monooxygenase, GAPDH, glyceraldehyde3-phosphate dehydrogenase.

The results of this study showed that the majority of enzymes in thekynurenine pathway and serotonin synthesis were expressed in hES cellsat four days after treatment of hES cells with 1 mM valproate (FIG. 7).Indoleamine 2, 3 dioxygenase INDO, catabolizes tryptophan into thekynurenine pathway, and produces kynurenine as an end product. Theexpression of tryptophan 2,3 dioxygenase (TDO or TDO2) was also examinedTDO2, like INDO, catalyzes the first step in the kynurenine pathway.These data suggested that TDO2 expression was upregulated in hES cellstreated with valproate in comparison to untreated controls. The ratelimiting enzyme in 5HT synthesis, TPH1, was also expressed in hES cells(FIG. 7). Expression of these enzymes supported the conclusion that hEScells recapitulate metabolic pathways of tryptophan catabolism andserotonin synthesis. Interestingly, VPA induced pronounced expression ofrate-limiting enzymes in this pathway.

Example 6 Developmental Toxicology Screening for Prenatal AlcoholExposure

To identify differentially secreted metabolites in response to alcohol,as well as the pathways involved in fetal alcohol syndrome, humanembryonic stem cells were treated with 0.0.1 and 0.3% ethanol for fourdays followed by LC/ESI-TOF mass spectrometry according to the generalmethods described above for valproate in Example 1. Extracellular mediawas collected and processed at 24 hours and four days after treatment,and 49,481 mass signals were detected following three technicalreplications. Of the 49,481 mass signals, 1,860 compounds weresignificantly different (p<0.05) in at least one treatment and had asignificant time change (<1 or >1). (Table 7). Binned masses wereannotated in silico by querying the neutral masses in several differentdatabases. These databases included Metlin, Biological MagneticResonance Data Bank (BMRB), NIST Chemistry WebBook, and the HumanMetabolome Database. A mass was considered identified when its neutralmass was within 10 ppm of a known compound annotated in one of thedatabases listed above.

The putative kynurenine compound (measured exact neutral mass 208.0816)was upregulated three-fold at day four, but not 24 hours, in bothtreatments (0.1%, p=0.001 and 0.3% p=0.002, respectively). Anotherputative metabolite in the kynurenine pathway, 8-methoxykynurenate(219.0532) was also upregulated at four days in response to both 0.1%and 0.3% alcohol treatment (p<0.05). The analysis also detected asignificant downregulation of 5-hydroxy-L-tryptophan (220.0848) at fourdays following 0.3% alcohol treatment (p<0.05) in comparison tountreated controls. 5-hydroxy-L-tryptophan is the only intermediatemetabolite between tryptophan and serotonin and its synthesis ismediated by tryptophan hydroxylase, the rate limiting enzyme inserotonin synthesis. These results suggest that alcohol exposure duringhuman development can affect serotonin bioavailability due toupregulation of tryptophan catabolism into kynurenines. In addition,alcohol exposure induced significant changes in metabolic pathways andsmall molecules involved in neural development such as glutamate,gabapentin, adrenaline and glutathione.

Example 7 Developmental Toxicology Screening of Neuronal Precursor Cells

Metabolomic assessment of teratogens on embryonic development is notlimited exclusively to hESCs. The methods of the invention are alsouseful with other progenitor stem cells, including lineage-restrictedstem cells such as neural precursor cells. To illustrate the efficacy oftoxicology screening on lineage-specific stem cells, neuronal precursorsderived from hESCs were treated with 1 mM valproate according to themethods described in Example 1.

Approximately 135 compounds were differentially secreted in VPA-treatedneuronal precursors versus control. (See Table 6). The results of thisstudy illustrated that the methods of the invention reveal alterationsin the metabolic profile of lineage-specific stem cells in response toteratogen exposure.

The results disclosed herein are set forth in the following tables

TABLE 3 Cellular metabolites measured in human embryonic stem cellstreated with 1 mM of valproate EXP RT roundMASS time trt Fold Probtannotation.1 annotation.2 1 mM 8.31910526 103.056358  4 D 1 mM1.987286671 0.01734377 gamma-Aminobutryic acid VPA VPA 1 mM 6.78779869103.098349  4 D 1 mM 2.143101233 0.00047552 2-Aminoisobutyric acid VPAVPA 1 mM 8.39854546 113.082093  4 D 1 mM 16.4054129 0.013426841-Pyrroline-5-carboxylic acid VPA VPA 1 mM 11.7534444 120.043328  4 D 1mM 1.355758298 0.03951319 3,4-Dihydroxybutyric acid VPA VPA 1 mM85.7330833 121.088708 24 H 1 mM 10.30519572 0.02442001 PhenylethylamineVPA VPA 1 mM 7.307128 122.071261  4 D 1 mM −2.2989897 0.01870947 UnknownVPA VPA 1 mM 38.1047857 129.070626  8 D 1 mM 3.023878137 0.039882132-Ketobutyric acid; 2- VPA VPA Oxobutyric acid; alpha- Ketobutyric acid;alpha- Ketobutyrate 1 mM 14.2761702 136.038753  8 D 1 mM 2.6967092810.00083818 Hypoxanthine Allopurinol VPA VPA 1 mM 31.3610896 141.114252 8 D 1 mM 1.865419366 0.02273706 Unknown VPA VPA 1 mM 43.9201842143.095482  8 D 1 mM 2.064797071 0.03120757 1- VPA VPAAminocyclohexanecarboxylic acid 1 mM 51.283453 144.113677 24 H 1 mM4.820891632 0.02775836 Caprylic acid Valproic acid VPA VPA 1 mM51.283453 144.113677  4 D 1 mM 8.26720694 0.0011089 Caprylic acidValproic acid VPA VPA 1 mM 16.307931 147.068314  4 D 1 mM −2.053806120.03872229 3-Methyloxindole VPA VPA 1 mM 16.307931 147.068314 24 H 1 mM−1.80875876 0.037812 3-Methyloxindole VPA VPA 1 mM 22.5095926 153.079352 4 D 1 mM −2.65737163 0.01268393 Dopamine VPA VPA 1 mM 5.36288806155.068364  8 D 1 mM −1.34957018 0.0476914 L-Histidine VPA VPA 1 mM20.6395854 155.072941  4 D 1 mM 3.82298622 0.00676609 L-Histidine VPAVPA 1 mM 14.2071091 160.060653  8 D 1 mM 12.348809 1.23E−06 Unknown VPAVPA 1 mM 14.3670392 161.081539  8 D 1 mM 4.777314979 0.0001252 UnknownVPA VPA 1 mM 44.8165285 162.067353  4 D 1 mM 1.640573238 0.01289447Unknown VPA VPA 1 mM 31.5154611 162.124096 24 H 1 mM 1.9112281390.01933211 Unknown VPA VPA 1 mM 31.3624074 165.079533  8 D 1 mM1.705269784 0.00206584 4-(3-Pyridyl)-butanoic acid VPA VPA 1 mM32.0426154 167.094942  8 D 1 mM −2.20640862 0.01874726 MethyldopamineVPA VPA 1 mM 20.0098065 173.083484  4 D 1 mM 5.458861144 0.001986322-Oxoarginine VPA VPA 1 mM 15.3496532 177.082617  8 D 1 mM 2.3977811710.01291216 Unknown VPA VPA 1 mM 24.1314286 179.094351  4 D 1 mM1.851635336 0.03464009 Salsolinol Homophenylalanine VPA VPA 1 mM21.8046482 187.064183  8 D 1 mM 2.839427352 0.00590774 Unknown VPA VPA 1mM 21.8046482 187.064183  4 D 1 mM 3.356831218 1.24E−05 Unknown VPA VPA1 mM 23.317 187.08492  4 D 1 mM 3.781608467 0.03987705 6-Acetamido-3-VPA VPA oxohexanoate 1 mM 27.7865556 189.042631  8 D 1 mM 2.8657246570.02214848 Kynurenic acid VPA VPA 1 mM 61.5462473 196.090684  4 D 1 mM3.519815968 0.01102697 Unknown VPA VPA 1 mM 24.0551398 197.105003  4 D 1mM 2.231478645 0.00076838 L-Metanephrine VPA VPA 1 mM 20.0989286197.175657  8 D 1 mM 4.237754463 0.00034728 Unknown VPA VPA 1 mM73.9582188 198.16015  4 D 1 mM 2.039619449 0.01232495 5-Dodecenoic acidVPA VPA 1 mM 29.2935714 201.100569  4 D 1 mM 5.966976107 0.00328699Unknown VPA VPA 1 mM 28.6036393 203.115212  8 D 1 mM 1.8725444950.00040874 L-Glutamic acid n-butyl ester Acetylcarnitine VPA VPA 1 mM9.07926923 209.06985  8 D 1 mM −12.4054314 0.018489574-Carboxyphenylglycine VPA VPA 1 mM 48.3434453 213.079468  8 D 1 mM2.512458907 0.02116099 Unknown VPA VPA 1 mM 44.6001887 214.064259  4 D 1mM 1.446032522 0.02767084 Unknown VPA VPA 1 mM 44.6001887 214.064259  8D 1 mM 1.783609761 0.00082206 Unknown VPA VPA 1 mM 69.6687917 214.06435624 H 1 mM 1.316493137 0.0171064 Unknown VPA VPA 1 mM 18.7504371216.094569  4 D 1 mM 2.349086763 0.01043169 Unknown VPA VPA 1 mM30.5773235 216.100485  4 D 1 mM 2.050108123 0.04358571 Unknown VPA VPA 1mM 6.39474737 218.076897  4 D 1 mM 1.737243521 0.02003307 Unknown VPAVPA 1 mM 6.68071429 219.144891 24 H 1 mM −1.58008262 0.02066251 UnknownVPA VPA 1 mM 10.5609423 220.085746  4 D 1 mM −2.39827983 1.63E−065-Hydroxytryptophan VPA VPA 1 mM 53.8814512 222.078039  4 D 1 mM2.882259036 0.02550855 Unknown VPA VPA 1 mM 73.0997018 223.049411  8 D 1mM −4.31392173 0.04819747 7,8-Dihydro-7,8- VPA VPA dihydroxykynurenate 1mM 6.45641791 229.095757  8 D 1 mM 2.4471457 0.00094873 MalonylcarnitineVPA VPA 1 mM 23.7927189 229.095855  4 D 1 mM 2.057653416 0.00038761Malonylcarnitine VPA VPA 1 mM 55.5371429 229.145042  4 D 1 mM2.032140286 0.00236036 Unknown VPA VPA 1 mM 19.9783175 229.164555  8 D 1mM 3.779774064 1.34E−08 Unknown VPA VPA 1 mM 32.6065714 229.201979  4 D1 mM 3.088058322 0.00439209 Unknown VPA VPA 1 mM 9.79255 230.080163  4 D1 mM −1.383766 0.02197836 Unknown VPA VPA 1 mM 7.80846914 233.123128  8D 1 mM 6.784300156 0.00043647 Unknown VPA VPA 1 mM 14.3537949 236.080213 4 D 1 mM −3.35334287 0.00032832 N′-Formylkynurenine VPA VPA 1 mM45.2867439 238.12327  8 D 1 mM 3.718961983 0.014668422-Amino-3-methylbutyric VPA VPA acid 1 mM 12.1171264 244.109135  4 D 1mM 1.659329044 0.01962434 Unknown VPA VPA 1 mM 12.127642 245.119507  4 D1 mM 2.061936638 0.02383097 Unknown VPA VPA 1 mM 19.8036863 246.100428 4 D 1 mM 4.924577653 0.02636633 N-Acetyl-D-tryptophan VPA VPA 1 mM8.947 247.140975  8 D 1 mM 2.877468231 0.01777412 Unknown VPA VPA 1 mM19.7590488 247.173942  8 D 1 mM 3.071620539 3.15E−06 Unknown VPA VPA 1mM 48.7837308 248.191881  4 D 1 mM 2.692786782 0.00724013 Unknown VPAVPA 1 mM 8.00665714 249.119037  4 D 1 mM 1.948008537 0.01865508 UnknownVPA VPA 1 mM 53.1723271 256.1093  8 D 1 mM 3.068428571 0.00024804D-2-Amino-3-hydroxybutyric gamma-Amino- VPA VPA acid beta-hydroxybutyricacid 1 mM 23.22678 257.099256  8 D 1 mM 2.601240877 0.000330495-Methylcytidine VPA VPA 1 mM 5.57045 257.891738 24 H 1 mM −1.170155290.01640996 Unknown VPA VPA 1 mM 7.08067539 258.019715 24 H 1 mM1.315125063 0.02206679 Unknown VPA VPA 1 mM 22.8759189 258.121153  4 D 1mM 1.510263204 0.01588551 Unknown VPA VPA 1 mM 28.8676889 258.133722 24H 1 mM 5.578974665 0.03000729 Unknown VPA VPA 1 mM 47.6525584 258.133727 4 D 1 mM −1.91733177 0.01442461 Unknown VPA VPA 1 mM 18.7499759259.11867  4 D 1 mM 1.504620863 0.02037249 N-(gamma-L- VPA VPAGlutamyl)amino-D-proline 1 mM 13.2221957 260.083648  8 D 1 mM2.984522231 0.02760558 Unknown VPA VPA 1 mM 22.373 264.109282  4 D 1 mM−1.74811488 0.01791457 Acetyl-N-formyl-5- VPA VPA methoxykynurenamine 1mM 58.8093824 265.132541  8 D 1 mM 2.363623094 0.03359569(2R,3S)-rel-2,3-dihydroxy-- VPA VPA Butanoic acid 1 mM 27.779593271.112691  4 D 1 mM 1.685060044 0.03867385 Unknown VPA VPA 1 mM24.7259575 272.124009  4 D 1 mM 2.036511555 0.02960407 Unknown VPA VPA 1mM 44.0582051 272.168272  8 D 1 mM 2.056227653 0.00325332 Unknown VPAVPA 1 mM 41.65612 272.211662  8 D 1 mM −5.12943527 0.006430513-Oxo-delta1-steroid VPA VPA 1 mM 39.378469 273.105953  4 D 1 mM2.674742484 0.00030929 Unknown VPA VPA 1 mM 8.93373171 276.136639  4 D 1mM 5.215118375 0.0006752 Unknown VPA VPA 1 mM 67.6599775 280.237772 24 H1 mM 2.086232575 0.04410145 Linoleic acid Octadecadienoic VPA VPA acid 1mM 14.2211017 281.125398  8 D 1 mM 8.170361997 1.56E−061-Methyladenosine VPA VPA 1 mM 71.5568571 282.225649  4 D 1 mM4.282045127 0.00101203 Unknown VPA VPA 1 mM 72.7757434 282.253397  8 D 1mM −1.86257691 0.03054583 Oleic acid Elaidic acid VPA VPA 1 mM59.4208378 284.19613  4 D 1 mM 5.253576839 0.0351483 Unknown VPA VPA 1mM 5.70108824 284.980449  4 D 1 mM 1.366608495 0.03819988 Unknown VPAVPA 1 mM 6.9018125 285.140063  4 D 1 mM 15.96344365 0.00293691 UnknownVPA VPA 1 mM 64.3362162 286.186749  4 D 1 mM 2.524154118 0.04701735N-Acetyl-leucyl-leucine VPA VPA 1 mM 64.3362162 286.186749 24 H 1 mM2.577549261 0.00532464 N-Acetyl-leucyl-leucine VPA VPA 1 mM 75.3938769288.263273  4 D 1 mM 2.556553115 0.0003234 Unknown VPA VPA 1 mM26.6263806 289.137569  8 D 1 mM 7.105814367 0.00077408 Unknown VPA VPA 1mM 15.0419293 289.139413  8 D 1 mM 3.953690666 0.00671585 Unknown VPAVPA 1 mM 15.8950204 295.128678  8 D 1 mM 2.286752138 1.23E−06N6,N6-Dimethyladenosine VPA VPA 1 mM 6.02850649 301.172858  4 D 1 mM5.302600282 0.00165331 Unknown VPA VPA 1 mM 59.5394364 301.222733  4 D 1mM 4.091131755 0.01328711 Unknown VPA VPA 1 mM 72.4551579 304.237816  8D 1 mM −5.09223853 0.00158305 Arachidonic acid VPA VPA 1 mM 44.6849231305.936181  8 D 1 mM 2.13864941 0.00372138 3-Iodo-4- VPA VPAhydroxyphenylpyruvate 1 mM 7.93489796 306.092269 24 H 1 mM −2.599078130.00116532 Unknown VPA VPA 1 mM 22.339 306.121765 24 H 1 mM 2.6660429080.00540921 Z-Gly-Pro; Z-Gly-Pro-OH VPA VPA 1 mM 59.461 306.180711  4 D 1mM 2.390810858 0.01473431 VPA VPA 1 mM 12.9788342 307.161748  8 D 1 mM5.76411852 0.00135244 Unknown VPA VPA 1 mM 4.76167568 308.158497  8 D 1mM 3.212062578 7.02E−05 Unknown VPA VPA 1 mM 7.58973529 316.131974  4 D1 mM 1.861931503 0.01887819 Unknown VPA VPA 1 mM 66.9950694 316.200989 4 D 1 mM 2.178145003 0.00392399 Gibberellin A12 aldehyde VPA VPA 1 mM62.665 319.244008 24 H 1 mM 3.088914632 0.0457215 Unknown VPA VPA 1 mM19.019754 320.137541  4 D 1 mM 2.083198045 0.04299189 Unknown VPA VPA 1mM 67.8541343 320.230187  4 D 1 mM 1.784846494 0.03271491 Unknown VPAVPA 1 mM 67.8541343 320.230187 24 H 1 mM 1.981647012 0.01061379 UnknownVPA VPA 1 mM 10.672 321.168775 24 H 1 mM 2.153375627 0.00361195 UnknownVPA VPA 1 mM 35.4656491 324.169472  4 D 1 mM 2.684214566 0.00585101Unknown VPA VPA 1 mM 63.932859 326.0008 24 H 1 mM 1.479797739 0.00340947Unknown VPA VPA 1 mM 63.932859 326.0008  4 D 1 mM 1.541142217 0.01010729Unknown VPA VPA 1 mM 62.4897344 328.242558  4 D 1 mM 1.8312134950.03531113 Docosahexaenoic acid VPA VPA 1 mM 55.092 329.001202 24 H 1 mM1.889887032 0.01315641 Unknown VPA VPA 1 mM 6.02840404 330.105879  8 D 1mM 4.856779538 0.01414085 Unknown VPA VPA 1 mM 12.8257065 330.153322  4D 1 mM −1.46094311 0.02278769 Unknown VPA VPA 1 mM 47.63185 330.240548 4 D 1 mM 2.777910272 0.01585359 Unknown VPA VPA 1 mM 67.7267647330.242694  4 D 1 mM 3.168939244 0.02399703 Unknown VPA VPA 1 mM9.01253333 331.103633  8 D 1 mM 5.010657754 0.00879812 Unknown VPA VPA 1mM 18.8430244 334.151446 24 H 1 mM 7.598422851 0.04853637 Unknown VPAVPA 1 mM 4.05694118 336.031706  4 D 1 mM −23.1557728 5.36E−05 UnknownVPA VPA 1 mM 6.7701658 336.15353  4 D 1 mM 2.062222503 0.01789166Unknown VPA VPA 1 mM 54.915974 347.982073  4 D 1 mM 16.748964510.02976287 Unknown VPA VPA 1 mM 45.6079091 348.203076  4 D 1 mM3.375263185 0.00190076 Unknown VPA VPA 1 mM 6.04629167 349.134979  8 D 1mM 1.449645356 0.02177991 Unknown VPA VPA 1 mM 67.3780816 352.221576 24H 1 mM 2.329951622 0.01190993 Prostaglandin VPA VPA 1 mM 22.8247245353.157641  4 D 1 mM 3.01822425 0.00974537 2-Keto-3-Methylvaleric acidVPA VPA 1 mM 19.103773 353.158931  4 D 1 mM 2.134354771 0.00286811Unknown VPA VPA 1 mM 29.94892 355.242828  4 D 1 mM 3.7515843610.01212028 Unknown VPA VPA 1 mM 15.1070313 356.156972  4 D 1 mM5.181249294 0.00024122 I-Glutamic-gamma- VPA VPA semialdehyde 1 mM59.1895507 358.229755  4 D 1 mM 4.389936283 0.00450968 Unknown VPA VPA 1mM 7.78296 359.071286  8 D 1 mM 3.504721971 0.02819084 Unknown VPA VPA 1mM 27.8286847 359.198793  4 D 1 mM 2.67566964 0.04513681 Unknown VPA VPA1 mM 7.67294845 362.15214 24 H 1 mM 3.677690313 0.01613594 Aminohexanoicacid VPA VPA 1 mM 6.16412 364.18324  4 D 1 mM 4.422922613 0.01590116Unknown VPA VPA 1 mM 19.3098372 364.185514  8 D 1 mM 2.5292330910.00135633 Gibberellin A44 VPA VPA 1 mM 53.9854054 366.239292  4 D 1 mM6.176116644 3.03E−05 3b-Allotetrahydrocortisol VPA VPA 1 mM 17.7238836372.188649  4 D 1 mM 3.32395304 2.43E−07 Ornithine VPA VPA 1 mM11.4481111 374.168865  8 D 1 mM 3.707636994 0.00715743 Unknown VPA VPA 1mM 13.8172619 374.207426  8 D 1 mM 2.50185816 0.03397986 Unknown VPA VPA1 mM 17.6750468 388.183189  8 D 1 mM 3.124878291 0.00060074 Malic acidDiglycolic acid VPA VPA 1 mM 21.3150329 392.209899  4 D 1 mM 2.4029389580.02146723 Unknown VPA VPA 1 mM 79.7277263 404.258123  4 D 1 mM2.231633324 0.02122436 7a,12a-Dihydroxy-3-oxo-4- VPA VPA cholenoic acid1 mM 24.7042427 407.206755  4 D 1 mM 2.564362115 0.03457095 Unknown VPAVPA 1 mM 16.9907536 408.172332  8 D 1 mM 1.47549599 0.01779219 4- VPAVPA Hydroxyphenylacetaldehyde; 1 mM 16.9907536 408.172332  4 D 1 mM1.84894204 0.00218606 4- VPA VPA Hydroxyphenylacetaldehyde; 1 mM29.02944 411.227331  4 D 1 mM 3.412904392 0.00663705 Gln His Lys VPA VPA1 mM 31.0764706 411.788303  4 D 1 mM 4.812211329 0.01959219 Unknown VPAVPA 1 mM 9.74277941 412.191819  8 D 1 mM 4.778970957 0.02280244 UnknownVPA VPA 1 mM 24.0870602 416.213608  8 D 1 mM 1.904483779 0.00013882Unknown VPA VPA 1 mM 13.7165 420.160178  4 D 1 mM 4.500233939 0.04166145Unknown VPA VPA 1 mM 27.4410904 421.219685  4 D 1 mM 2.929998650.01453378 Unknown VPA VPA 1 mM 84.9993429 424.278966  4 D 1 mM2.302178983 0.02079967 1b,3a,7a,12a-Tetrahydroxy- VPA VPA 5b-cholanoicacid 1 mM 84.9993429 424.278966 24 H 1 mM 2.318995467 0.000169021b,3a,7a,12a-Tetrahydroxy- VPA VPA 5b-cholanoic acid 1 mM 54.5975206429.099036  4 D 1 mM 3.524698852 2.05E−05 Unknown VPA VPA 1 mM25.5684706 430.183077  4 D 1 mM 1.148698355 0.00012401 Unknown VPA VPA 1mM 47.39725 432.071275  4 D 1 mM 2.645059178 0.01175067 Unknown VPA VPA1 mM 14.5288987 445.217914  8 D 1 mM 2.820595921 0.00824753 Unknown VPAVPA 1 mM 7.5585 445.286693  4 D 1 mM −1.13705339 0.04453994 Unknown VPAVPA 1 mM 19.9357624 455.226453  8 D 1 mM 2.768491323 0.0146344 AdipateVPA VPA 1 mM 84.7522195 470.350048  4 D 1 mM 3.767741534 0.00027941Unknown VPA VPA 1 mM 8.479 471.146232 24 H 1 mM 2.569343893 0.0259874210-Formyldihydrofolate VPA VPA 1 mM 22.7650396 471.202698  4 D 1 mM2.257302866 1.30E−06 Unknown VPA VPA 1 mM 15.4262845 491.253192  8 D 1mM 4.916392167 0.00321994 Unknown VPA VPA 1 mM 60.5202059 493.458959  4D 1 mM 2.789487333 0.00131052 Unknown VPA VPA 1 mM 44.8878444 502.216027 4 D 1 mM 1.922921676 0.00968802 Unknown VPA VPA 1 mM 14.5675854502.227438  8 D 1 mM 3.101787817 0.00862582 Unknown VPA VPA 1 mM31.0262667 504.2848  4 D 1 mM 5.119489655 7.48E−05 Unknown VPA VPA 1 mM17.4495833 516.244788  4 D 1 mM 2.722628233 0.00035163 Unknown VPA VPA 1mM 44.14925 527.321492  8 D 1 mM 2.213454933 0.00740287 Unknown VPA VPA1 mM 70.8363171 528.362659  4 D 1 mM 2.941801698 0.03554601 Unknown VPAVPA 1 mM 74.25245 530.344375 24 H 1 mM 3.680750602 0.03848341 UnknownVPA VPA 1 mM 9.15167857 532.249475  8 D 1 mM 7.170133597 0.00736475Unknown VPA VPA 1 mM 29.9863488 535.254098  8 D 1 mM 6.0490140010.00097203 Unknown VPA VPA 1 mM 87.9394 535.392963  4 D 1 mM 1.801251960.03183737 Unknown VPA VPA 1 mM 8.02928261 549.20135  8 D 1 mM11.08087574 0.00035145 Unknown VPA VPA 1 mM 19.5000458 550.228302  4 D 1mM 2.35969435 0.00064579 Unknown VPA VPA 1 mM 24.7983934 551.248871 24 H1 mM 1.396388132 0.01890342 Unknown VPA VPA 1 mM 74.3730889 552.32624424 H 1 mM 1.885569072 0.031072 Lithocholate 3-O-glucuronide VPA VPA 1 mM75.3072222 561.322428  4 D 1 mM 5.181249294 0.00798648 Unknown VPA VPA 1mM 14.1881491 565.230107  4 D 1 mM 2.651300141 0.04312251 Unknown VPAVPA 1 mM 5.97658065 574.262774  8 D 1 mM 2.982867719 0.00682514 UnknownVPA VPA 1 mM 70.4439429 594.37144  4 D 1 mM 2.591881931 0.049706742-Hydroxyadenine VPA VPA 1 mM 15.895551 598.283549  8 D 1 mM 4.0747173850.00446383 2-Hydroxyadenine VPA VPA 1 mM 76.5242159 599.574322  8 D 1 mM2.569165805 2.63E−05 Unknown VPA VPA 1 mM 76.2647 600.576755  4 D 1 mM1.998614186 0.00464652 Unknown VPA VPA 1 mM 76.2647 600.576755  8 D 1 mM2.690920931 0.00059418 Unknown VPA VPA 1 mM 79.7894576 613.589997  8 D 1mM 3.099853425 0.01314731 Unknown VPA VPA 1 mM 8.59329167 658.254492  8D 1 mM 13.32441233 0.02367066 Unknown VPA VPA 1 mM 60.8503 688.51026  4D 1 mM 3.690969971 0.00301918 Unknown VPA VPA 1 mM 69.7080175 690.409258 4 D 1 mM 3.89061979 0.001728 Unknown VPA VPA 1 mM 65.32996 738.583348 4 D 1 mM 3.877159268 0.00021681 Unknown VPA VPA 1 mM 69.7792917810.640892  4 D 1 mM 3.934008296 0.00141534 Unknown VPA VPA 1 mM21.6729286 921.002586 24 H 1 mM 10.91318268 0.00761215 Unknown VPA VPA 1mM 5.86856 1007.84992  4 D 1 mM 23.49041018 0.043240093-Dehydrocarnitine VPA VPA

TABLE 4 Cellular metabolites produced in hESCs treated with 22 μMvalproate cpdID RT MASSavg time _trt Fold P-value Compound 1 Compound277 28.21 99.0681 4 days VPA −1.81 0.020 N-Methyl-2-pyrrolidinone 10312.00 103.0991 4 days VPA −2.24 0.028 Gamma-Aminobutryic acid2-Aminoisobutyric acid 141 34.03 113.0840 4 days VPA −1.43 0.013 Unknown189 12.08 119.0473 8 days VPA 1.22 0.040 4-Amino-3-hydroxybutanoate 21096.44 120.0436 4 days VPA −4.22 0.006 3,4-Dihydroxybutyric acid 26319.93 129.0426 4 days VPA −1.27 0.021 Pyroglutamic acid1-Pyrroline-4-hydroxy-2- carboxylate 323 29.83 134.0939 4 days VPA −1.430.034 Unknown 329 16.96 136.0384 24 hours VPA −2.09 0.038 HypoxanthineAllopurinol 343 12.98 141.0412 4 days VPA −1.24 0.0111,4,4,6-Tetrahydro-6- 2-Aminomuconate oxonicotinate semialdehyde 36211.40 141.9381 4 days VPA 1.19 0.034 Unknown 396 11.97 146.0683 4 daysVPA −1.02 0.002 Glutamine 413 44.84 148.0638 8 days VPA −2.72 0.004Unknown 444 12.37 144.0687 8 days VPA −1.42 0.014 Unknown 449 20.19146.0066 8 days VPA −1.24 0.002 2,4-dicarboxylic acid 496 30.40 161.06888 days VPA −1.23 0.019 4-Methyl-L-glutamate 2,2′-Iminodipropanoate 43124.83 164.4009 4 days VPA −1.17 0.049 Unknown 603 72.83 173.9844 8 daysVPA −1.80 0.017 Unknown 604 20.34 174.0160 4 days VPA −1.36 0.003cis-Aconitate Dehydroascorbate 636 42.18 178.0994 8 days VPA 1.47 0.002Phenylvaleric acid 646 24.00 181.0740 4 days VPA −1.11 0.004 SalsolinolHomophenylalanine 671 24.90 187.0609 4 days VPA −1.40 0.018 Unknown 67436.08 187.0973 4 days VPA 2.14 0.042 Unknown 812 29.93 204.0899 8 daysVPA 1.08 0.034 L-Tryptophan 843 24.94 208.0840 4 days VPA −1.14 0.004Kynurenine Formyl-4-hydroxykynurenamine 893 44.64 214.1680 8 days VPA−2.11 0.005 Fenamic acid 1089 47.40 242.0808 8 days VPA 1.87 0.02Unknown 1104 7.98 243.9760 4 days VPA 1.92 0.032 Unknown 1282 44.30274.0947 8 days VPA 1.78 0.019 3-Oxo-delta4-steroid 1447 43.40 300.27848 days VPA −2.22 0.033 Unknown 1440 27.94 314.2032 4 day VPA −1.12 0.012Unknown 1637 24.91 330.1480 4 day VPA −1.42 0.004 Unknown 1684 11.94336.1634 4 days VPA −1.13 0.023 Unknown 1691 34.87 338.0974 4 days VPA1.74 0.033 Unknown 1776 39.67 342.1130 4 day VPA 1.46 0.044 Unknown 181624.40 348.1139 8 days VPA 2.56 0.025 Unknown 1838 11.00 361.9194 4 daysVPA −1.08 0.004 Unknown 1948 12.00 384.1664 8 days VPA 1.38 0.018Unknown 1949 14.98 387.1498 4 days VPA −1.26 0.001 Unknown 2084 64.24414.2934 4 days VPA −1.20 0.031 Unknown 2131 88.14 426.2983 4 days VPA1.85 0.022 Cholanoic acid 2134 74.20 427.1200 8 day VPA −1.88 0.044Unknown 2138 26.91 428.2423 8 day VPA 1.70 0.003 Unknown 2144 34.14431.2733 4 days VPA −1.24 0.041 Unknown 2186 32.68 441.1394 8 days VPA−1.16 0.022 Folate Folic acid 2191 64.67 442.2934 8 days VPA 1.79 0.001Unknown 2214 92.89 440.3448 8 days VPA −1.84 0.037 Unknown 2233 12.00444.0841 8 days VPA −1.30 0.041 Unknown 2244 64.41 449.3198 24 hours VPA−1.78 0.024 Unknown 2291 87.74 470.3249 4 days VPA −0.18 0.002 Unknown2244 30.91 467.2631 8 days VPA 1.69 0.004 Unknown 743 13.81 197.0186 8day VPA −1.39 0.016 Unknown 636 42.18 178.0994 8 days 1.47 0.010 Unknown

TABLE 6 Cellular metabolites measured in neural precursors derived fromhESells treated with 1 mM of valproate EXP RT roundMASS time trt Foldannotation.1 annotation.2 NS 1 mM NS 1 mM VPA 36.648 102.0322438 2 d VPA−2.23119 2-Ketobutyric acid Acetoacetic acid NS 1 mM NS 1 mM VPA 36.648102.0322438 4 d VPA −1.83846 2-Ketobutyric acid Acetoacetic acid NS 1 mM9.33225 119.958645 4 d NS 1 mM 6.98086 Unknown VPA VPA NS 1 mM 12.2841121.0621387 4 d NS 1 mM 3.502341 Unknown VPA VPA NS 1 mM 24.10558125.0838833 2 d NS 1 mM 1.79316 Unknown VPA VPA NS 1 mM 30.43985125.08394 2 d NS 1 mM 1.529012 1-Methylhistamine VPA VPA NS 1 mM30.43985 125.08394 4 d NS 1 mM 1.576622 VPA VPA 1-Methylhistamine NS 1mM 23.33772 129.0573222 2 d NS 1 mM 1.543375 Pyroglutamic acid VPA VPANS 1 mM 23.33772 129.0573222 4 d NS 1 mM 1.663008 Pyroglutamic acid VPAVPA NS 1 mM 12.13216 131.0941359 4 d NS 1 mM 1.577796 L-IsoleucineAminocaproic acid VPA VPA NS 1 mM 12.13216 131.0941359 2 d NS 1 mM2.474877 L-Isoleucine Aminocaproic acid VPA VPA NS 1 mM 8.881211136.0366263 2 d NS 1 mM 2.287439 Erythronic acid Erythronic acid VPA VPANS 1 mM 8.881211 136.0366263 4 d NS 1 mM 2.653537 Erythronic acidErythronic acid VPA VPA NS 1 mM 12.00967 136.0376917 2 d NS 1 mM2.346054 Erythronic acid Erythronic acid VPA VPA NS 1 mM 12.00967136.0376917 4 d NS 1 mM 2.914393 Erythronic acid Erythronic acid VPA VPANS 1 mM 3.9669 138.04396 2 d NS 1 mM 1.521407 Urocanic acid NicotinamideN- VPA VPA oxide NS 1 mM 3.9669 138.04396 4 d NS 1 mM 2.642558 Urocanicacid Nicotinamide N- VPA VPA oxide NS 1 mM 4.28225 141.9392625 2 d NS 1mM 1.077336 5,10-Methylenetetrahydrofolate VPA VPA NS 1 mM 4.28225141.9392625 4 d NS 1 mM 1.111532 5,10-Methylenetetrahydrofolate VPA VPANS 1 mM 23.33784 143.0734947 2 d NS 1 mM 1.728349 Unknown VPA VPA NS 1mM 23.33784 143.0734947 4 d NS 1 mM 1.986515 Unknown VPA VPA NS 1 mM55.5845 144.1153313 4 d NS 1 mM 10.83178 Caprylic acid Valproic acid VPAVPA NS 1 mM 55.5845 144.1153313 2 d NS 1 mM 11.64535 Caprylic acidValproic acid VPA VPA NS 1 mM 5.609182 145.1572818 2 d NS 1 mM 1.00993Spermidine VPA VPA NS 1 mM 5.609182 145.1572818 4 d NS 1 mM 1.117314Spermidine VPA VPA NS 1 mM 33.6357 148.03738 4 d NS 1 mM −5.75294Citramalic acid Hydroxyglutaric VPA VPA acid NS 1 mM 33.6357 148.03738 2d NS 1 mM −1.49356 Citramalic acid Hydroxyglutaric VPA VPA acid NS 1 mM62.42614 152.1201762 4 d NS 1 mM 2.50602 Unknown VPA VPA NS 1 mM62.42614 152.1201762 2 d NS 1 mM 3.371426 Unknown VPA VPA NS 1 mM8.862333 158.0177333 2 d NS 1 mM 1.596402 Unknown VPA VPA NS 1 mM8.862333 158.0177333 4 d NS 1 mM 2.236076 Unknown VPA VPA NS 1 mM8.269857 158.1374571 4 d NS 1 mM 3.106829 Unknown VPA VPA NS 1 mM8.269857 158.1374571 2 d NS 1 mM 3.626498 Unknown VPA VPA NS 1 mM10.07033 159.0688667 2 d NS 1 mM −2.87026 Indoleacetaldehyde VPA VPA NS1 mM 10.07033 159.0688667 4 d NS 1 mM −1.35298 Indoleacetaldehyde VPAVPA NS 1 mM 12.85888 161.0509118 2 d NS 1 mM 2.601443 Unknown VPA VPA NS1 mM 12.85888 161.0509118 4 d NS 1 mM 5.136057 Unknown VPA VPA NS 1 mM6.713565 166.0840609 4 d NS 1 mM 33.80296 Unknown VPA VPA NS 1 mM6.713565 166.0840609 2 d NS 1 mM 90.97629 Unknown VPA VPA NS 1 mM23.58909 168.0687909 2 d NS 1 mM 4.649885 Unknown VPA VPA NS 1 mM23.58909 168.0687909 4 d NS 1 mM 5.02165 Unknown VPA VPA NS 1 mM31.08471 171.1250706 4 d NS 1 mM 1.626943 Unknown VPA VPA NS 1 mM62.57554 172.1454 4 d NS 1 mM 1.745543 Capric acid Decanoic acid VPA VPANS 1 mM 62.57554 172.1454 2 d NS 1 mM 1.8794 Capric acid Decanoic acidVPA VPA NS 1 mM 20.68721 175.0830857 2 d NS 1 mM 1.153935N-Carboxyethyl-gamma- VPA VPA aminobutyric acid NS 1 mM 20.68721175.0830857 4 d NS 1 mM 2.294392 N-Carboxyethyl-gamma- VPA VPAaminobutyric acid NS 1 mM 41.71109 176.0946 2 d NS 1 mM −1.60014Serotonin VPA VPA NS 1 mM 41.71109 176.0946 4 d NS 1 mM −1.23797Serotonin VPA VPA NS 1 mM 25.29 177.0469231 2 d NS 1 mM 3.379693N-Formyl-L-methionine VPA VPA NS 1 mM 25.29 177.0469231 4 d NS 1 mM3.82485 N-Formyl-L-methionine VPA VPA NS 1 mM 26.75621 177.0789684 2 dNS 1 mM 1.26513 5-Hydroxytryptophol VPA VPA NS 1 mM 26.75621 177.07896844 d NS 1 mM 1.423219 5-Hydroxytryptophol VPA VPA NS 1 mM 8.503333177.113375 2 d NS 1 mM −6.74695 Unknown VPA VPA NS 1 mM 8.503333177.113375 4 d NS 1 mM −2.88736 Unknown VPA VPA NS 1 mM 27.53982179.0938118 2 d NS 1 mM −2.71897 Salsolinol Homophenylalanine VPA VPA NS1 mM 27.53982 179.0938118 4 d NS 1 mM −1.71231 SalsolinolHomophenylalanine VPA VPA NS 1 mM 55.15089 179.0949632 4 d NS 1 mM−1.64525 Salsolinol Homophenylalanine VPA VPA NS 1 mM 55.15089179.0949632 2 d NS 1 mM −1.39458 Salsolinol Homophenylalanine VPA VPA NS1 mM 37.08443 185.1406571 4 d NS 1 mM −2.39254 Unknown VPA VPA NS 1 mM23.33726 187.0635211 2 d NS 1 mM 1.674013 Unknown VPA VPA NS 1 mM23.33726 187.0635211 4 d NS 1 mM 1.921765 Unknown VPA VPA NS 1 mM28.77111 187.1206333 2 d NS 1 mM 2.457813 8-Amino-7-oxononanoic acid VPAVPA NS 1 mM 28.77111 187.1206333 4 d NS 1 mM 3.5593618-Amino-7-oxononanoic acid VPA VPA NS 1 mM 62.50765 190.1720118 4 d NS 1mM 1.758553 Unknown VPA VPA NS 1 mM 62.50765 190.1720118 2 d NS 1 mM1.940004 Unknown VPA VPA NS 1 mM 7.850167 196.0933333 4 d NS 1 mM1.937281 Unknown VPA VPA NS 1 mM 7.850167 196.0933333 2 d NS 1 mM6.390957 Unknown VPA VPA NS 1 mM 45.36418 197.1060727 2 d NS 1 mM1.280472 L-Metanephrine VPA VPA NS 1 mM 45.36418 197.1060727 4 d NS 1 mM1.62879 L-Metanephrine VPA VPA NS 1 mM 8.363925 199.0952975 2 d NS 1 mM10.44025 Unknown VPA VPA NS 1 mM 8.363925 199.0952975 4 d NS 1 mM10.88407 Unknown VPA VPA NS 1 mM 22.22829 206.06375 4 d NS 1 mM 2.263839Unknown VPA VPA NS 1 mM 22.22829 206.06375 2 d NS 1 mM 4.317383 UnknownVPA VPA NS 1 mM 62.46678 208.1829217 4 d NS 1 mM 2.052914 Unknown VPAVPA NS 1 mM 62.46678 208.1829217 2 d NS 1 mM 2.734885 Unknown VPA VPA NS1 mM 9.2636 211.0349075 4 d NS 1 mM 1.516795 Creatine phosphate VPA VPANS 1 mM 9.2636 211.0349075 2 d NS 1 mM 1.893074 Creatine phosphate VPAVPA NS 1 mM 44.11333 212.1400167 4 d NS 1 mM −9.35257 Unknown VPA VPA NS1 mM 44.11333 212.1400167 2 d NS 1 mM −6.85493 Unknown VPA VPA NS 1 mM7.746115 217.1048885 2 d NS 1 mM 2.916422 N-a-Acetylcitrulline VPA VPANS 1 mM 7.746115 217.1048885 4 d NS 1 mM 23.86569 N-a-AcetylcitrullineVPA VPA NS 1 mM 22.26729 217.1307097 2 d NS 1 mM 2.122093Propionylcarnitine VPA VPA NS 1 mM 22.26729 217.1307097 4 d NS 1 mM2.236406 Propionylcarnitine VPA VPA NS 1 mM 16.1278 220.0841 2 d NS 1 mM−1.25214 5-Hydroxytryptophan 5-Hydroxy-L- VPA VPA tryptophan NS 1 mM16.1278 220.0841 4 d NS 1 mM 1.215413 5-Hydroxytryptophan 5-Hydroxy-L-VPA VPA tryptophan NS 1 mM 9.809786 220.0845 2 d NS 1 mM −1.061025-Hydroxytryptophan 5-Hydroxy-L- VPA VPA tryptophan NS 1 mM 9.809786220.0845 4 d NS 1 mM 1.371126 5-Hydroxytryptophan 5-Hydroxy-L- VPA VPAtryptophan NS 1 mM 12.15958 220.0845895 2 d NS 1 mM −1.542755-Hydroxytryptophan 5-Hydroxy-L- VPA VPA tryptophan NS 1 mM 12.15958220.0845895 4 d NS 1 mM 1.162644 5-Hydroxytryptophan 5-Hydroxy-L- VPAVPA tryptophan NS 1 mM 8.4172 223.92951 2 d NS 1 mM −3.24844 Unknown VPAVPA NS 1 mM 8.4172 223.92951 4 d NS 1 mM −2.85631 Unknown VPA VPA NS 1mM 22.009 225.62685 4 d NS 1 mM 2.716119 Unknown VPA VPA NS 1 mM 22.009225.62685 2 d NS 1 mM 3.854852 Unknown VPA VPA NS 1 mM 10.0963 227.019384 d NS 1 mM 1.631698 L-Glutamic acid 5-phosphate VPA VPA NS 1 mM 6.0771227.09052 4 d NS 1 mM −5.41292 Deoxycytidine VPA VPA NS 1 mM 6.0771227.09052 2 d NS 1 mM −2.98012 Deoxycytidine VPA VPA NS 1 mM 14.51476228.05894 4 d NS 1 mM 3.339111 Unknown VPA VPA NS 1 mM 14.51476228.05894 2 d NS 1 mM 6.425869 Unknown VPA VPA NS 1 mM 67.13919230.1515667 4 d NS 1 mM −5.02528 Dodecanedioic acid VPA VPA NS 1 mM67.13919 230.1515667 2 d NS 1 mM −2.98776 Dodecanedioic acid VPA VPA NS1 mM 19.28286 234.1010143 2 d NS 1 mM −1.25569 5-Methoxytryptophan VPAVPA NS 1 mM 19.28286 234.1010143 4 d NS 1 mM −1.188695-Methoxytryptophan VPA VPA NS 1 mM 10.51438 236.0815625 4 d NS 1 mM1.367658 N′-Formylkynurenine VPA VPA NS 1 mM 17.826 238.0864167 2 d NS 1mM 5.397657 VPA VPA Propanoic acid NS 1 mM 17.826 238.0864167 4 d NS 1mM 5.703771 VPA VPA Propanoic acid NS 1 mM 7.8115 239.087425 4 d NS 1 mM1.950568 Unknown VPA VPA NS 1 mM 7.8115 239.087425 2 d NS 1 mM 30.57925Unknown VPA VPA NS 1 mM 42.05716 246.1469838 4 d NS 1 mM −2.288013-Hydroxydodecanedioic acid VPA VPA NS 1 mM 42.05716 246.1469838 2 d NS1 mM −1.71537 3-Hydroxydodecanedioic acid VPA VPA NS 1 mM 30.669256.09664 4 d NS 1 mM 1.692487 Aryl beta-D-glucoside VPA VPA NS 1 mM30.669 256.09664 2 d NS 1 mM 1.966122 Aryl beta-D-glucoside VPA VPA NS 1mM 59.82681 256.1080938 2 d NS 1 mM 2.962348 Unknown VPA VPA NS 1 mM59.82681 256.1080938 4 d NS 1 mM 3.845884 Unknown VPA VPA NS 1 mM69.57173 258.18226 4 d NS 1 mM 4.075358 Tetradecanedioic acid VPA VPA NS1 mM 69.57173 258.18226 2 d NS 1 mM 5.744182 Tetradecanedioic acid VPAVPA NS 1 mM 22.0274 264.11209 2 d NS 1 mM 1.230997 Acetyl-N-formyl-5-VPA VPA methoxykynurenamine NS 1 mM 22.0274 264.11209 4 d NS 1 mM1.338241 Acetyl-N-formyl-5- VPA VPA methoxykynurenamine NS 1 mM 11.94583268.0806 4 d NS 1 mM 3.240011 3-Deoxy-D-glycero-D-galacto-2- VPA VPAnonulosonic acid NS 1 mM 11.94583 268.0806 2 d NS 1 mM 3.3298153-Deoxy-D-glycero-D-galacto-2- VPA VPA nonulosonic acid NS 1 mM 20.58271270.1203286 2 d NS 1 mM 1.808333 L-gamma-Glutamyl-L-hypoglycin; VPA VPANS 1 mM 20.58271 270.1203286 4 d NS 1 mM 1.890677L-gamma-Glutamyl-L-hypoglycin; VPA VPA NS 1 mM 56.5351 272.08566 4 d NS1 mM 1.178071 5-S-Cysteinyldopamine VPA VPA NS 1 mM 56.5351 272.08566 2d NS 1 mM 1.718998 5-S-Cysteinyldopamine VPA VPA NS 1 mM 64.11407278.0251024 4 d NS 1 mM 5.17024 Unknown VPA VPA NS 1 mM 64.11407278.0251024 2 d NS 1 mM 6.667641 Unknown VPA VPA NS 1 mM 28.90879290.1501643 4 d NS 1 mM 3.329013 Unknown VPA VPA NS 1 mM 28.90879290.1501643 2 d NS 1 mM 8.490919 Unknown VPA VPA NS 1 mM 26.42889295.1063913 2 d NS 1 mM 7.265582 Unknown VPA VPA NS 1 mM 26.42889295.1063913 4 d NS 1 mM 8.896581 Unknown VPA VPA NS 1 mM 73.28533315.2406111 4 d NS 1 mM 2.599877 Decanoylcarnitine VPA VPA NS 1 mM73.28533 315.2406111 2 d NS 1 mM 3.899691 Decanoylcarnitine VPA VPA NS 1mM 77.38144 318.2193688 4 d NS 1 mM 1.932263 Leukotriene A4 VPA VPA NS 1mM 77.38144 318.2193688 2 d NS 1 mM 2.342543 Leukotriene A4 VPA VPA NS 1mM 41.33024 324.1144588 4 d NS 1 mM 3.317923 Acetohexamide VPA VPA NS 1mM 41.33024 324.1144588 2 d NS 1 mM 3.713445 Acetohexamide VPA VPA NS 1mM 33.60576 330.1013765 4 d NS 1 mM 2.25561 Unknown VPA VPA NS 1 mM33.60576 330.1013765 2 d NS 1 mM 2.310447 Unknown VPA VPA NS 1 mM35.06233 331.1049867 4 d NS 1 mM 2.719086 Unknown VPA VPA NS 1 mM35.06233 331.1049867 2 d NS 1 mM 3.041317 Unknown VPA VPA NS 1 mM 52.557349.22592 4 d NS 1 mM 2.41993 Unknown VPA VPA NS 1 mM 52.557 349.22592 2d NS 1 mM 6.652089 Unknown VPA VPA NS 1 mM 53.57858 350.2096 4 d NS 1 mM1.508076 Prostaglandin E3 VPA VPA NS 1 mM 53.57858 350.2096 2 d NS 1 mM1.612834 Prostaglandin E3 VPA VPA NS 1 mM 65.76353 356.2702895 4 d NS 1mM 2.05875 Tetracosahexaenoic acid VPA VPA NS 1 mM 65.76353 356.27028952 d NS 1 mM 2.405825 Tetracosahexaenoic acid VPA VPA NS 1 mM 81.79271369.2880824 4 d NS 1 mM 6.636435 cis-5-Tetradecenoylcarnitine VPA VPA NS1 mM 81.79271 369.2880824 2 d NS 1 mM 9.965816cis-5-Tetradecenoylcarnitine VPA VPA NS 1 mM 27.34076 374.1222235 4 d NS1 mM 2.300022 Unknown VPA VPA NS 1 mM 27.34076 374.1222235 2 d NS 1 mM2.889783 Unknown VPA VPA NS 1 mM 8.881 380.164 4 d NS 1 mM 2.519949Unknown VPA VPA NS 1 mM 8.881 380.164 2 d NS 1 mM 2.633667 Unknown VPAVPA NS 1 mM 22.46583 385.1025667 4 d NS 1 mM 1.45497S-Inosyl-L-homocysteine VPA VPA NS 1 mM 22.46583 385.1025667 2 d NS 1 mM1.533705 S-Inosyl-L-homocysteine VPA VPA NS 1 mM 68.31689 386.23145 4 dNS 1 mM 6.386163 1-tridecanoyl-sn-glycero-3- VPA VPA phosphate NS 1 mM68.31689 386.23145 2 d NS 1 mM 7.117538 1-tridecanoyl-sn-glycero-3- VPAVPA phosphate NS 1 mM 89.1946 390.27608 2 d NS 1 mM 1.6828557-Hydroxy-3-oxocholanoic acid VPA VPA NS 1 mM 89.1946 390.27608 4 d NS 1mM 3.296512 7-Hydroxy-3-oxocholanoic acid VPA VPA NS 1 mM 26.42904394.2127308 2 d NS 1 mM 2.670186 Unknown VPA VPA NS 1 mM 26.42904394.2127308 4 d NS 1 mM 3.50273 unknown VPA VPA NS 1 mM 76.06971398.2439857 4 d NS 1 mM 2.845315 Unknown VPA VPA NS 1 mM 76.06971398.2439857 2 d NS 1 mM 3.609136 Unknown VPA VPA NS 1 mM 33.53038399.2100125 2 d NS 1 mM 6.345173 unknown VPA VPA NS 1 mM 33.53038399.2100125 4 d NS 1 mM 8.777833 unknown VPA VPA NS 1 mM 8.9305406.1058875 4 d NS 1 mM 1.976577 unknown VPA VPA NS 1 mM 8.9305406.1058875 2 d NS 1 mM 2.00634 unknown VPA VPA NS 1 mM 65.76447409.3155632 4 d NS 1 mM 4.662331 Unknown VPA VPA NS 1 mM 65.76447409.3155632 2 d NS 1 mM 5.930421 Unknown VPA VPA NS 1 mM 18.93053416.0834333 4 d NS 1 mM 3.527468 Unknown VPA VPA NS 1 mM 18.93053416.0834333 2 d NS 1 mM 4.202864 Unknown VPA VPA NS 1 mM 8.6127416.20208 2 d NS 1 mM 3.29783 Lactone VPA VPA NS 1 mM 8.6127 416.20208 4d NS 1 mM 3.495266 Lactone VPA VPA NS 1 mM 14.963 420.05275 2 d NS 1 mM−3.434 Unknown VPA VPA NS 1 mM 14.963 420.05275 4 d NS 1 mM −3.09195Unknown VPA VPA NS 1 mM 62.93221 427.1025357 2 d NS 1 mM 3.882014Unknown VPA VPA NS 1 mM 62.93221 427.1025357 4 d NS 1 mM 6.045912Unknown VPA VPA NS 1 mM 33.59771 430.1185143 2 d NS 1 mM 3.469797N-Ethylmaleimide-S-glutathione VPA VPA NS 1 mM 33.59771 430.1185143 4 dNS 1 mM 3.98295 N-Ethylmaleimide-S-glutathione VPA VPA NS 1 mM 24.9718434.19845 4 d NS 1 mM 3.713915 Unknown VPA VPA NS 1 mM 24.9718 434.198452 d NS 1 mM 3.76882 Unknown VPA VPA NS 1 mM 23.07 434.1985875 2 d NS 1mM 2.416021 Unknown VPA VPA NS 1 mM 23.07 434.1985875 4 d NS 1 mM3.44524 Unknown VPA VPA NS 1 mM 37.33089 438.1460579 2 d NS 1 mM2.829002 Unknown VPA VPA NS 1 mM 37.33089 438.1460579 4 d NS 1 mM3.253909 Unknown VPA VPA NS 1 mM 5.410909 441.9424 4 d NS 1 mM −3.5345Unknown VPA VPA NS 1 mM 5.410909 441.9424 2 d NS 1 mM −2.45689 UnknownVPA VPA NS 1 mM 34.58505 443.2362947 2 d NS 1 mM 2.648862 Unknown VPAVPA NS 1 mM 34.58505 443.2362947 4 d NS 1 mM 3.427563 Unknown VPA VPA NS1 mM 33.85267 445.1694 2 d NS 1 mM −1.31487 Tetrahydrofolic acidTetrahydrofolate VPA VPA NS 1 mM 33.85267 445.1694 4 d NS 1 mM −1.04073Tetrahydrofolic acid Tetrahydrofolate VPA VPA NS 1 mM 38.63717449.1638333 2 d NS 1 mM 3.04704 Unknown VPA VPA NS 1 mM 38.63717449.1638333 4 d NS 1 mM 4.206864 Unknown VPA VPA NS 1 mM 21.9772456.2448 4 d NS 1 mM 4.145738 unknown VPA VPA NS 1 mM 21.9772 456.2448 2d NS 1 mM 8.390862 unknown VPA VPA NS 1 mM 42.40369 467.1731077 2 d NS 1mM −10.3323 Unknown VPA VPA NS 1 mM 42.40369 467.1731077 4 d NS 1 mM−4.42263 Unknown VPA VPA NS 1 mM 23.41189 474.1090778 4 d NS 1 mM2.094815 Unknown VPA VPA NS 1 mM 23.41189 474.1090778 2 d NS 1 mM2.928466 Unknown VPA VPA NS 1 mM 22.666 482.1554563 4 d NS 1 mM 2.075721Unknown VPA VPA NS 1 mM 22.666 482.1554563 2 d NS 1 mM 2.519832 UnknownVPA VPA NS 1 mM 75.55014 493.3252405 4 d NS 1 mM 2.09415 VPA VPA1-(9E-hexadecenoyl)-sn-glycero- 3-phosphocholine NS 1 mM 75.55014493.3252405 2 d NS 1 mM 2.34894 VPA VPA 1-(9E-hexadecenoyl)-sn-glycero-3-phosphocholine NS 1 mM 37.34533 506.1856556 2 d NS 1 mM 2.031088Unknown VPA VPA NS 1 mM 37.34533 506.1856556 4 d NS 1 mM 2.405509unknown VPA VPA NS 1 mM 23.67792 514.162208 2 d NS 1 mM 2.2744 unknownVPA VPA NS 1 mM 23.67792 514.162208 4 d NS 1 mM 3.27837 unknown VPA VPANS 1 mM 36.44195 514.2430667 4 d NS 1 mM 4.332807 Unknown VPA VPA NS 1mM 36.44195 514.2430667 2 d NS 1 mM 4.565629 Unknown VPA VPA NS 1 mM41.29621 527.3552786 2 d NS 1 mM 9.44893 Unknown VPA VPA NS 1 mM41.29621 527.3552786 4 d NS 1 mM 12.3251 Unknown VPA VPA NS 1 mM21.94381 534.2784846 4 d NS 1 mM 2.818876 unknown VPA VPA NS 1 mM21.94381 534.2784846 2 d NS 1 mM 2.912693 unknown VPA VPA NS 1 mM26.05061 546.3146929 2 d NS 1 mM 1.574871 Unknown VPA VPA NS 1 mM26.05061 546.3146929 4 d NS 1 mM 3.130802 Unknown VPA VPA NS 1 mM25.92929 556.13945 4 d NS 1 mM 4.837329 unknown VPA VPA NS 1 mM 25.92929556.13945 2 d NS 1 mM 5.293236 unknown VPA VPA NS 1 mM 9.093727575.1451545 4 d NS 1 mM 2.795937 Unknown VPA VPA NS 1 mM 9.093727575.1451545 2 d NS 1 mM 4.232054 Unknown VPA VPA NS 1 mM 36.92372575.3159222 2 d NS 1 mM 2.816601 Unknown VPA VPA NS 1 mM 36.92372575.3159222 4 d NS 1 mM 3.446719 unknown VPA VPA NS 1 mM 80.9297583.44194 2 d NS 1 mM 1.503812 Unknown VPA VPA NS 1 mM 80.9297 583.441944 d NS 1 mM 4.532225 Unknown VPA VPA NS 1 mM 4.824 594.2327 4 d NS 1 mM2.212473 Unknown VPA VPA NS 1 mM 4.824 594.2327 2 d NS 1 mM 4.279664Unknown VPA VPA NS 1 mM 41.278 632.232825 2 d NS 1 mM 3.171377 UnknownVPA VPA NS 1 mM 41.278 632.232825 4 d NS 1 mM 4.764468 Unknown VPA VPANS 1 mM 14.97571 659.1506941 4 d NS 1 mM 3.054219 Unknown VPA VPA NS 1mM 23.39707 660.1513786 2 d NS 1 mM 3.542683 Unknown VPA VPA NS 1 mM23.39707 660.1513786 4 d NS 1 mM 4.100844 Unknown VPA VPA NS 1 mM 14.884682.2853 2 d NS 1 mM 2.823221 Unknown VPA VPA NS 1 mM 14.884 682.2853 4d NS 1 mM 5.153298 Unknown VPA VPA NS 1 mM 33.64471 822.2805429 2 d NS 1mM 2.668941 Unknown VPA VPA NS 1 mM 33.64471 822.2805429 4 d NS 1 mM3.816104 Unknown VPA VPA NS 1 mM 83.24742 907.54555 4 d NS 1 mM 1.507945Unknown VPA VPA NS 1 mM 83.24742 907.54555 2 d NS 1 mM 1.586431 UnknownVPA VPA NS 1 mM 31.5316 908.22015 2 d NS 1 mM 1.640148 Unknown VPA VPANS 1 mM 31.5316 908.22015 4 d NS 1 mM 1.998983 Unknown VPA VPA NS 1 mM33.44333 1028.3246 2 d NS 1 mM 5.833998 Unknown VPA VPA NS 1 mM 33.443331028.3246 4 d NS 1 mM 8.654592 Unknown VPA VPA NS 1 mM 4.6491251291.75965 2 d NS 1 mM 1.739338 beta-D-Glucosyl-1,4-N-acetyl-D- VPA VPAglucosaminyldiphosphoundecaprenol NS 1 mM 4.649125 1291.75965 4 d NS 1mM 1.793695 beta-D-Glucosyl-1,4-N-acetyl-D- VPA VPAglucosaminyldiphosphoundecaprenol

TABLE 7 Cellular metabolites measured in hES cells treated with alcoholRetention Experiment time Mass Time Fold p-value Compound 1 Compound 2ETOH 0.1 15.48433 99.0689 4 D 1.434154 0.034571 N-Methyl-2-pyrrolidinoneETOH 0.1 52.01225 99.1043 4 D 2.703447 0.012638 Unknown ETOH 0.113.40565 120.2112 4 D 4.847027 0.029776 Unknown ETOH 0.1 16.73904129.0452 24 H 1.502328 0.002871 3,4-Dihydroxybutyric acid ETOH 0.188.64043 130.9541 24 H 1.631614 0.046779 Unknown ETOH 0.1 22.22892131.0746 24 H −1.85703 0.037466 3-Methylindole ETOH 0.1 14.35336 131.0764 D 3.62907 0.014778 Unknown ETOH 0.1 3.958833 148.0052 24 H −1.948410.034059 Unknown ETOH 0.1 52.88652 148.016 4 D −2.44409 0.0471222-Oxo-4-methylthiobutanoic acid ETOH 0.1 19.18355 168.0434 4 D −1.467850.019426 Homogentisic acid Vanillic acid ETOH 0.1 26.70635 171.1244 24 H2.376107 0.008413 GABA analogue ETOH 0.1 22.17997 187.1343 24 H 1.487820.045452 (+/−)-2-(4′- Isobutylphenyl)propionitrile ETOH 0.1 46.31086187.1348 4 D 2.329144 4.21E−05 (+/−)-2-(4′- Isobutylphenyl)propionitrileETOH 0.1 5.935143 194.073 4 D −1.38924 0.003217 Phenanthrene-9,10-oxideETOH 0.1 31.19917 195.124 4 D −2.22499 0.004386 Benzenemethanol, 2-(2-a-[1- aminopropoxy)-3-methyl- (ethylamino)ethyl]-p- hydroxy-Benzylalcohol ETOH 0.1 38.99212 195.1253 4 D 2.158606 0.00953 Benzenemethanol,2-(2- a-[1- aminopropoxy)-3-methyl- (ethylamino)ethyl]-p- hydroxy-Benzylalcohol ETOH 0.1 48.37093 197.1769 4 D −3.11904 0.016197 Unknown ETOH0.1 9.675726 203.1138 4 D −1.70728 0.023636 Acetylcarnitine L-Glutamicacid n- butyl ester ETOH 0.1 6.747279 205.1304 4 D −1.2578 0.005318Pantothenol dimethylbutanamide ETOH 0.1 36.18938 210.0922 4 D 1.8642560.040527 3-(2,5-Dimethoxy phenylpropionic acid ETOH 0.1 24.52067229.0949 24 H −2.33044 0.008877 Malonylcarnitine ETOH 0.1 17.7027243.1089 4 D −2.18071 0.016141 Unknown ETOH 0.1 64.66999 266.1613 24 H−1.51898 0.047652 Unknown ETOH 0.1 42.3656 268.2487 4 D −1.549710.015019 Unknown ETOH 0.1 4.86619 271.9364 24 H 2.629339 0.04245 UnknownETOH 0.1 43.99398 272.16 24 H 1.929598 0.032191 Unknown ETOH 0.143.99398 272.16 4 D 2.186768 0.003018 Unknown ETOH 0.1 63.00428 285.228524 H 4.002774 0.018095 Unknown ETOH 0.1 37.97297 292.1862 4 D 2.2393810.0496 Unknown ETOH 0.1 4.014175 293.9773 4 D 1.938848 0.036775 UnknownETOH 0.1 6.160071 294.0957 24 H 1.269183 0.005089 Unknown ETOH 0.18.967061 295.1521 4 D 1.513407 0.042025 Unknown ETOH 0.1 71.55535296.2308 24 H 3.545035 0.003758 Unknown ETOH 0.1 50.75387 298.174 4 D−3.00237 0.045113 Unknown ETOH 0.1 18.88505 300.1147 4 D 2.6862620.023485 Unknown ETOH 0.1 17.18696 300.1656 24 H 1.548853 0.036582Unknown ETOH 0.1 7.719471 301.1345 4 D 6.648828 0.030822 Unknown ETOH0.1 15.22357 312.1341 4 D −2.01503 0.041156 Unknown ETOH 0.1 26.51229315.6732 4 D 2.014609 0.010998 Unknown ETOH 0.1 18.82373 325.2711 4 D−1.75625 0.013853 Unknown ETOH 0.1 20.94557 325.2714 4 D −2.074260.00333 Unknown ETOH 0.1 8.542672 337.2012 24 H 1.402499 0.000372Unknown ETOH 0.1 3.85935 353.2765 4 D 2.622059 0.002006 Unknown ETOH 0.125.16993 357.1781 4 D 2.217294 0.001346 Unknown ETOH 0.1 24.01428359.1532 4 D 1.535704 0.033 Unknown ETOH 0.1 18.50245 360.1321 4 D2.11023 0.004465 Unknown ETOH 0.1 83.72506 362.2787 24 H 2.8198140.04795 Unknown ETOH 0.1 83.72506 362.2787 4 D 2.916423 0.023844 UnknownETOH 0.1 27.98054 368.2122 4 D 1.69537 0.02565 Unknown ETOH 0.1 20.76168379.1771 24 H −1.72285 0.021135 Unknown ETOH 0.1 20.76168 379.1771 4 D1.539861 0.019592 Unknown ETOH 0.1 15.20829 383.1721 4 D 1.9145430.043581 Unknown ETOH 0.1 23.38956 384.2127 4 D −2.55567 0.025704Unknown ETOH 0.1 51.65871 386.1724 4 D 5.308852 0.032774 (+)-EudesminETOH 0.1 19.97914 387.0812 4 D −1.97698 0.024641 Unknown ETOH 0.119.97914 387.0812 24 H 1.739051 0.018391 Unknown ETOH 0.1 17.53242388.1815 24 H −1.44894 0.012322 Unknown ETOH 0.1 46.346 388.2349 4 D1.946524 0.002762 Unknown ETOH 0.1 15.90129 393.1889 24 H −1.430980.022977 Unknown ETOH 0.1 6.259963 396.1687 24 H 1.717607 0.047952Unknown ETOH 0.1 51.66325 403.1978 4 D 3.11601 0.048224 Unknown ETOH 0.130.70733 405.2001 4 D 2.668076 0.001676 Unknown ETOH 0.1 16.21743408.1636 4 D 1.965641 0.028858 Unknown ETOH 0.1 21.14975 417.2386 4 D−1.97972 0.007183 Unknown ETOH 0.1 33.09057 417.2338 4 D 2.0325630.016902 Unknown ETOH 0.1 26.77212 420.1862 4 D 3.282511 0.030236Unknown ETOH 0.1 18.03482 429.2533 4 D −1.80751 0.006213 Unknown ETOH0.1 30.24237 429.2535 4 D 1.804876 0.044332 Unknown ETOH 0.1 35.58196431.2501 4 D 1.532408 0.037928 Unknown ETOH 0.1 32.18393 437.2042 24 H24.53212 0.001124 Unknown ETOH 0.1 4.808947 440.0223 4 D −1.527850.03034 Unknown ETOH 0.1 24.13915 443.2381 4 D 2.985557 0.023682 UnknownETOH 0.1 67.0705 443.3216 4 D 1.751997 0.037074 Unknown ETOH 0.151.58546 444.2237 4 D 2.130512 0.031074 Unknown ETOH 0.1 33.51823460.9391 4 D −4.51805 0.005949 Unknown ETOH 0.1 22.95657 462.2217 24 H−1.98563 0.0437 Unknown ETOH 0.1 25.3287 464.225 24 H −1.63071 0.025852Unknown ETOH 0.1 46.51446 467.3804 4 D 2.068091 0.042613 Unknown ETOH0.1 51.6158 468.2002 4 D 1.875012 0.015762 Unknown ETOH 0.1 30.37291471.1928 4 D 2.001387 0.022662 glucuronide ETOH 0.1 30.72707 471.7804 4D −2.83569 0.037476 Unknown ETOH 0.1 30.28867 478.2761 4 D 1.6988990.000475 Unknown ETOH 0.1 72.7735 482.3062 24 H −1.95925 0.042853Unknown ETOH 0.1 6.676207 485.2069 24 H −2.01419 0.013732 Unknown ETOH0.1 66.73744 487.3472 4 D 2.931014 0.007475 Unknown ETOH 0.1 21.72729489.2127 4 D −1.51037 0.0314 Unknown ETOH 0.1 31.22083 510.8202 4 D2.488196 0.011267 Unknown ETOH 0.1 34.35986 521.9924 24 H −1.445930.032994 Unknown ETOH 0.1 34.57864 525.3161 4 D 1.551324 0.037545Unknown ETOH 0.1 51.73057 526.2773 4 D 3.445707 0.006877 Unknown ETOH0.1 23.87065 530.314 4 D 1.964552 0.006366 L-Oleandrosyl-oleandolideETOH 0.1 32.50661 531.2876 4 D 2.106138 0.024689 Unknown ETOH 0.135.58454 531.3191 4 D −1.25162 0.027019 Unknown ETOH 0.1 66.31491531.3736 4 D 3.862674 0.01116 Unknown ETOH 0.1 32.24719 541.3274 4 D2.161601 0.038319 Unknown ETOH 0.1 17.6573 545.3029 4 D −1.394840.043629 Unknown ETOH 0.1 31.891 554.8471 4 D 2.038489 0.037688 UnknownETOH 0.1 15.78741 555.2406 4 D 1.835025 0.014923 Unknown ETOH 0.15.742094 555.8505 4 D −1.28922 0.017625 Unknown ETOH 0.1 88.02533556.3971 4 D −3.11839 0.016192 Unknown ETOH 0.1 31.97996 559.8329 4 D−1.7213 0.049678 Unknown ETOH 0.1 24.7039 574.3397 4 D 1.58436 0.02116Unknown ETOH 0.1 31.71105 574.3427 4 D 1.750055 0.026643 Unknown ETOH0.1 47.90467 576.096 4 D 1.361314 0.021201 Unknown ETOH 0.1 16.90923577.2825 24 H 1.727637 0.047154 Unknown ETOH 0.1 35.26918 589.6938 4 D1.819573 0.027966 Unknown ETOH 0.1 31.54325 591.3789 4 D 1.5782220.000714 Unknown ETOH 0.1 25.14927 596.3543 4 D 1.395808 0.049214L-Urobilinogen; ETOH 0.1 32.67372 603.3535 4 D −2.62952 0.015253 UnknownETOH 0.1 8.056862 612.1509 24 H −1.47366 0.02889 Oxidized glutathioneOxidized glutathione; Glutathione disulfide; GSSG; Oxiglutatione ETOH0.1 33.68866 619.3409 4 D 1.856648 0.030722 Unknown ETOH 0.1 32.73907620.8861 4 D 2.248558 0.00893 Unknown ETOH 0.1 32.08734 635.4065 4 D1.392618 0.030885 Unknown ETOH 0.1 5.903429 646.7084 4 D −1.271470.020652 Unknown ETOH 0.1 26.7452 661.3846 4 D 1.295402 0.046573 UnknownETOH 0.1 33.48766 677.9101 24 H −2.22083 0.028347 Unknown ETOH 0.131.11764 693.4124 4 D 2.318353 0.028562 Unknown ETOH 0.1 28.2045695.4286 24 H −32.9384 0.027453 Unknown ETOH 0.1 33.70266 699.9225 4 D1.719036 0.039493 Unknown ETOH 0.1 40.84822 702.2497 4 D −2.769450.001031 Neu5Acalpha2-3Galbeta1- Neu5Acalpha2- 4Glcbeta-Sp6Galbeta1-4Glcbeta-Sp ETOH 0.1 34.62439 707.3928 4 D 2.136871 0.0353Unknown ETOH 0.1 56.18269 707.4296 24 H 1.489574 0.046762 Unknown ETOH0.1 33.73428 708.9387 4 D 2.159654 0.006959 Unknown ETOH 0.1 4.826824711.8344 24 H −3.02053 0.013448 Unknown ETOH 0.1 33.89008 730.4494 4 D2.613893 0.009133 Unknown ETOH 0.1 47.76987 731.0954 4 D −2.775790.004811 Unknown ETOH 0.1 5.918027 732.007 4 D 1.795393 0.021782 UnknownETOH 0.1 35.028 751.4193 4 D 1.87008 0.032616 Unknown ETOH 0.1 34.12668752.4629 4 D 2.066515 0.031345 Unknown ETOH 0.1 69.32865 765.5211 24 H1.873064 0.033127 Unknown ETOH 0.1 34.33545 774.4767 4 D 2.1036580.020363 Unknown ETOH 0.1 89.29926 774.5055 4 D −2.40077 0.006755Unknown ETOH 0.1 5.886782 780.241 4 D −1.31403 0.025516 Unknown ETOH 0.134.52749 796.4891 4 D 1.926524 0.049615 Unknown ETOH 0.1 34.60124796.9917 4 D 1.818186 0.015806 Unknown ETOH 0.1 4.613879 820.8181 4 D−1.47009 0.047535 Unknown ETOH 0.1 5.259716 888.8041 4 D −1.457710.021932 Unknown ETOH 0.1 8.502051 909.5934 24 H 2.274264 0.037836Unknown ETOH 0.1 5.217833 913.8074 24 H −1.86064 0.028059 Unknown ETOH0.1 5.399211 921.0025 4 D 1.677834 0.001526 Unknown ETOH 0.1 3.646902994.0917 24 H 1.441829 0.019979 Unknown ETOH 0.1 3.705141 1008.072 4 D1.30378 0.048393 Unknown ETOH 0.1 5.177162 1038.786 4 D 1.6778340.030851 Unknown ETOH 0.3 85.57399 83.0372 24 H 2.472036 0.010882Unknown ETOH 0.3 15.48433 99.0689 4 D 1.337742 0.043286N-Methyl-2-pyrrolidinone ETOH 0.3 15.48433 99.0689 24 H 1.4678450.043638 N-Methyl-2-pyrrolidinone ETOH 0.3 52.01225 99.1043 24 H3.331103 0.000378 Unknown ETOH 0.3 10.21225 101.1201 24 H 2.1919270.043209 Hexylamine ETOH 0.3 4.032816 111.9839 4 D −8.67642 0.018374Thiosulfate ETOH 0.3 3.767232 120.0436 4 D 1.936297 0.0345853,4-Dihydroxybutyric acid ETOH 0.3 13.40565 120.2112 4 D 3.900070.046375 Unknown ETOH 0.3 16.73904 129.0452 24 H 1.795891 3.32E−053,4-Dihydroxybutyric acid ETOH 0.3 88.64043 130.9541 24 H 2.0249690.006982 Unknown ETOH 0.3 22.22892 131.0746 4 D 2.502205 0.0498333-Methylindole ETOH 0.3 14.35336 131.076 4 D 4.050219 0.020549 UnknownETOH 0.3 3.958833 148.0052 24 H −1.72967 0.043053 Unknown ETOH 0.37.479235 149.0511 4 D −1.30477 0.014313 Amino-4methylthiobutyric acidETOH 0.3 27.80141 153.0811 24 H −1.6976 0.025771 Unknown ETOH 0.35.559732 155.0681 4 D 1.637846 0.022817 L-Histidine 4-propionic acidETOH 0.3 14.22357 161.0805 24 H −6.43527 0.032474 Unknown ETOH 0.344.88033 162.0662 4 D 1.799131 0.037703 Unknown ETOH 0.3 23.6763167.0941 24 H −2.83 0.012984 3-Methoxytyramine Phenylephrine ETOH 0.319.18355 168.0434 4 D 1.281914 0.028006 Homogentisic acid Vanillic acidETOH 0.3 26.70635 171.1244 24 H 3.755227 0.001253 GABA analogue ETOH 0.320.23014 173.084 24 H −1.43983 0.019446 1,3-Dimethyl-8-isoquinolinolETOH 0.3 28.52393 178.5546 24 H −2.76389 0.001401 Unknown ETOH 0.35.935143 194.073 4 D −1.39988 0.002956 Phenanthrene-9,10-oxide9-Hydroxyphenanthrene; 9-Phenanthrol ETOH 0.3 22.61355 194.0836 24 H−1.70303 0.030955 Unknown ETOH 0.3 31.19917 195.124 4 D −1.860130.015911 Benzenemethanol, 2-(2- a-[1- aminopropoxy)-3-methyl-(ethylamino)ethyl]-p- hydroxy-Benzyl alcohol ETOH 0.3 19.48063 201.17094 D −2.97874 0.009458 Unknown ETOH 0.3 6.747279 205.1304 4 D −1.338580.014168 Pantothenol dimethylbutanamide; ETOH 0.3 36.18938 210.0922 4 D1.849968 0.042032 3-(2,5-Dimethoxy phenylpropionic acid ETOH 0.3 6.62669218.0762 4 D −1.77129 0.047105 Unknown ETOH 0.3 27.57188 222.0401 24 H−2.11155 0.040386 Unknown ETOH 0.3 13.6845 223.119 24 H −2.739670.024694 Unknown Unknown ETOH 0.3 24.52067 229.0949 4 D −1.740380.010344 Malonylcarnitine Malonylcarnitine ETOH 0.3 55.50731 229.1457 4D −1.53336 0.028395 Unknown ETOH 0.3 32.9941 229.2025 24 H −1.376970.03113 Unknown ETOH 0.3 47.0879 234.125 24 H −1.63184 0.0291695-Methoxytryptophan ETOH 0.3 53.26863 234.1253 4 D −1.62383 0.0262915-Methoxytryptophan ETOH 0.3 3.673694 237.0041 24 H 2.989077 0.011016Unknown ETOH 0.3 5.176232 239.9592 24 H 1.640005 0.018097 Unknown ETOH0.3 27.39631 243.11 4 D −1.49547 0.024259 Unknown ETOH 0.3 6.626769247.1049 4 D −4.47566 0.039425 Unknown ETOH 0.3 9.0276 247.1408 4 D−2.75089 0.000424 Unknown ETOH 0.3 18.84552 256.1066 4 D −2.140730.02288 5-Ethyl-5-(1-methyl-3- carboxypropyl)barbituric acid ETOH 0.340.75868 267.2543 4 D −1.6481 0.029485 Unknown ETOH 0.3 40.75868267.2543 24 H −1.49599 0.021245 Unknown ETOH 0.3 42.3656 268.2487 4 D−1.51708 0.030774 Unknown ETOH 0.3 4.86619 271.9364 24 H 4.0696370.02742 Unknown ETOH 0.3 22.86183 275.1193 24 H −2.23674 0.044504Unknown ETOH 0.3 5.69776 284.9798 4 D −1.2223 0.026683 Unknown ETOH 0.314.88092 286.1519 4 D −1.86167 0.033393 Unknown ETOH 0.3 75.76147288.2632 24 H 1.96905 0.001271 Unknown ETOH 0.3 66.86661 293.1952 4 D−2.15382 0.004487 Unknown ETOH 0.3 4.014175 293.9773 4 D −2.267180.013307 Unknown ETOH 0.3 20.67831 294.1535 4 D −1.42237 0.008416Unknown ETOH 0.3 24.21651 294.1531 24 H −1.89159 0.02267 Unknown ETOH0.3 8.967061 295.1521 4 D 1.467032 0.021323 Unknown ETOH 0.3 66.35884298.1537 24 H 3.758351 0.016783 Unknown ETOH 0.3 19.66398 299.1929 24 H−1.57058 0.041283 Unknown ETOH 0.3 7.719471 301.1345 4 D 2.6782670.046654 Unknown ETOH 0.3 4.954547 303.8875 24 H 2.069669 0.049847Unknown ETOH 0.3 44.09424 313.199 24 H 2.291989 0.005146 Unknown ETOH0.3 26.51229 315.6732 4 D −2.86533 0.000573 Unknown ETOH 0.3 23.90107322.1166 4 D −2.4215 0.010241 Unknown ETOH 0.3 30.19245 324.1666 24 H−1.59251 0.037632 Unknown ETOH 0.3 20.72194 332.1367 4 D −2.077 0.009795Unknown ETOH 0.3 8.542672 337.2012 24 H 1.287435 0.004465 Unknown ETOH0.3 5.033976 340.9252 24 H −1.83604 0.037709 Unknown ETOH 0.3 5.033976340.9252 4 D 2.624968 0.049915 Unknown ETOH 0.3 68.02448 342.1482 24 H−1.85279 0.043066 Unknown ETOH 0.3 3.85935 353.2765 4 D 4.9041390.000229 Unknown ETOH 0.3 18.50245 360.1321 24 H −2.28881 0.005116Unknown ETOH 0.3 83.72506 362.2787 24 H −2.54153 0.005266 Unknown ETOH0.3 20.16372 365.1606 4 D −1.92519 0.027055 Unknown ETOH 0.3 11.47109375.1898 4 D 2.131693 0.028486 Unknown ETOH 0.3 26.07722 375.1886 4 D−4.75123 0.000746 Unknown ETOH 0.3 41.28494 378.2956 24 H 1.6364850.027631 Unknown ETOH 0.3 15.20829 383.1721 4 D 3.377369 0.002214Unknown ETOH 0.3 51.65871 386.1724 4 D 4.85106 0.031786 (+)-Eudesmin(+)-Eudesmin ETOH 0.3 19.97914 387.0812 4 D −1.69949 0.015257 UnknownETOH 0.3 46.346 388.2349 4 D −1.53209 0.040255 Unknown ETOH 0.3 15.90129393.1889 24 H −1.51089 0.011717 Unknown ETOH 0.3 19.69092 393.1886 4 D−2.05894 0.011258 Unknown ETOH 0.3 6.259963 396.1687 4 D 2.4220020.013544 Unknown ETOH 0.3 17.27421 403.1984 4 D −2.30266 0.00281 UnknownETOH 0.3 21.14975 417.2386 4 D −1.57309 0.03368 Unknown ETOH 0.333.09057 417.2338 4 D −1.77978 0.026439 Unknown ETOH 0.3 13.35295420.0513 4 D −1.90198 0.003417 Unknown ETOH 0.3 27.46219 421.2201 4 D−2.27332 0.012803 Unknown ETOH 0.3 18.03482 429.2533 4 D −2.200910.002807 Unknown ETOH 0.3 54.46495 440.0284 24 H 2.51037 0.011972Unknown ETOH 0.3 30.87201 443.2339 4 D 2.171362 0.0186 Unknown ETOH 0.367.0705 443.3216 4 D 1.688451 0.048544 Unknown ETOH 0.3 51.58546444.2237 24 H 2.241866 0.044444 Unknown ETOH 0.3 51.58546 444.2237 4 D2.030169 0.032667 Unknown ETOH 0.3 30.05687 444.2789 24 H −1.835030.021764 Unknown ETOH 0.3 54.8719 446.0431 24 H −2.22145 0.049006Unknown ETOH 0.3 54.8719 446.0431 4 D 1.933882 0.047398 Unknown ETOH 0.329.86415 447.2509 4 D −1.85819 0.025964 Unknown ETOH 0.3 29.86415447.2509 24 H 2.10322 0.036254 Unknown ETOH 0.3 30.45343 449.2653 4 D−2.41429 0.016795 Unknown ETOH 0.3 44.98056 449.2611 24 H −3.471360.038457 Unknown ETOH 0.3 28.27806 455.2052 24 H −2.50898 0.005338Unknown ETOH 0.3 33.51823 460.9391 4 D −3.60151 0.010558 Unknown ETOH0.3 22.95657 462.2217 4 D 1.935626 0.03474 Unknown ETOH 0.3 33.5733463.2914 24 H −1.66521 0.034862 Unknown ETOH 0.3 25.3287 464.225 24 H−1.71665 0.033532 Unknown ETOH 0.3 30.46172 466.2921 24 H −1.959380.012601 Unknown ETOH 0.3 33.68894 466.615 24 H −3.27865 0.008212Unknown ETOH 0.3 46.51446 467.3804 24 H −2.13465 0.013009 Unknown ETOH0.3 51.6158 468.2002 4 D 1.919859 0.003122 Unknown ETOH 0.3 30.37291471.1928 4 D 2.725649 0.008164 glucuronide ETOH 0.3 30.72707 471.7804 4D −3.44069 0.010281 Unknown ETOH 0.3 30.28867 478.2761 4 D 1.5099490.018484 Unknown ETOH 0.3 10.82859 482.1942 24 H −1.52795 0.033711Unknown ETOH 0.3 72.7735 482.3062 24 H −2.61806 0.0161 Unknown ETOH 0.310.8217 485.204 24 H 2.038065 0.038466 Unknown ETOH 0.3 66.73744487.3472 4 D 2.877867 0.020235 Unknown ETOH 0.3 30.83472 488.305 24 H−2.18525 0.009407 Unknown ETOH 0.3 30.88032 488.8071 24 H −1.919590.040672 Unknown ETOH 0.3 21.72729 489.2127 4 D 2.372158 0.000369Unknown ETOH 0.3 13.78553 502.2258 4 D 1.866325 0.010364 Unknown ETOH0.3 18.36887 505.2616 4 D 2.008892 0.036368 Unknown ETOH 0.3 5.891069509.6704 4 D 1.467845 0.0228 Unknown ETOH 0.3 31.20706 510.3182 24 H−2.26373 0.006715 Unknown ETOH 0.3 31.22083 510.8202 24 H −2.045140.041219 Unknown ETOH 0.3 31.22083 510.8202 4 D 2.502378 0.010461Unknown ETOH 0.3 46.57666 518.3914 4 D 2.288814 0.002533 Unknown ETOH0.3 46.57666 518.3914 24 H −2.70682 0.017765 Unknown ETOH 0.3 34.35986521.9924 24 H −1.58403 0.024454 Unknown ETOH 0.3 31.53434 523.8187 24 H−2.17272 0.02888 Unknown ETOH 0.3 51.73057 526.2773 4 D 2.7145250.010415 Unknown ETOH 0.3 71.36012 528.3631 4 D 2.361003 0.026297Unknown ETOH 0.3 23.87065 530.314 4 D 2.211921 0.001298L-Oleandrosyl-oleandolide ETOH 0.3 32.50661 531.2876 4 D 2.360840.014947 Unknown ETOH 0.3 35.58454 531.3191 4 D −3.0409 0.000281 UnknownETOH 0.3 66.31491 531.3736 4 D 2.87388 0.031879 Unknown ETOH 0.331.58889 532.8335 24 H −2.16926 0.015971 Unknown ETOH 0.3 51.52268539.4374 24 H −1.92987 0.031525 Unknown ETOH 0.3 32.24719 541.3274 24 H−2.12255 0.01445 Unknown ETOH 0.3 31.8519 554.3444 24 H −2.36953 0.00655Unknown ETOH 0.3 31.891 554.8471 24 H −2.29708 0.010734 Unknown ETOH 0.315.78741 555.2406 4 D 2.470837 0.001092 Unknown ETOH 0.3 5.742094555.8505 4 D −1.3396 0.023948 Unknown ETOH 0.3 5.742094 555.8505 24 H−1.51803 0.031838 Unknown ETOH 0.3 88.02533 556.3971 4 D −2.383860.035829 Unknown ETOH 0.3 31.97996 559.8329 4 D −2.56596 0.002155Unknown ETOH 0.3 14.9927 566.2265 4 D 1.624054 0.006806 Unknown ETOH 0.324.7039 574.3397 4 D 1.523934 0.011558 Unknown ETOH 0.3 47.90467 576.0964 D 1.557573 0.020904 Unknown ETOH 0.3 32.15777 576.3582 24 H −2.208860.012421 Unknown ETOH 0.3 16.90923 577.2825 4 D −8.91726 0.02781 UnknownETOH 0.3 5.903169 579.2519 4 D −2.11184 0.001075 Ethanesulfonic acidETOH 0.3 5.903169 579.2519 24 H −1.48021 0.01083 Ethanesulfonic acidETOH 0.3 31.54325 591.3789 24 H −1.98165 0.010883 Unknown ETOH 0.325.14927 596.3543 4 D 1.545421 0.015277 L-Urobilinogen; ETOH 0.325.14927 596.3543 24 H 1.834898 0.021555 L-Urobilinogen; ETOH 0.332.67372 603.3535 4 D −2.93997 0.007904 Unknown ETOH 0.3 56.3513611.4952 24 H −1.9811 0.007557 Unknown ETOH 0.3 4.936704 611.8727 4 D2.009588 0.018619 Unknown ETOH 0.3 32.70236 611.871 24 H −2.192080.044039 Unknown ETOH 0.3 8.056862 612.1509 24 H −1.59538 0.010611Oxidized glutathione ETOH 0.3 33.68866 619.3409 4 D 2.125939 0.015211Unknown ETOH 0.3 32.73907 620.8861 24 H −2.32076 0.039661 Unknown ETOH0.3 32.08734 635.4065 4 D 1.394744 0.031645 Unknown ETOH 0.3 32.95522642.397 24 H −2.21146 0.035834 Unknown ETOH 0.3 5.903429 646.7084 4 D2.946495 2.66E−05 Unknown ETOH 0.3 8.787827 658.2544 4 D −3.209610.030138 Unknown ETOH 0.3 26.7452 661.3846 4 D 1.546707 0.010714 UnknownETOH 0.3 16.3338 666.3836 24 H −1.37135 0.037278 Unknown ETOH 0.333.48766 677.9101 24 H −2.64653 0.009907 Unknown ETOH 0.3 40.84822702.2497 24 H 1.283337 0.019733 Neu5Acalpha2-3Galbeta1- 4Glcbeta-Sp ETOH0.3 40.84822 702.2497 4 D −2.21591 0.004389 Neu5Acalpha2-3Galbeta1-4Glcbeta-Sp ETOH 0.3 30.43889 707.3933 24 H −1.79166 0.048232 UnknownETOH 0.3 56.18269 707.4296 24 H −2.43361 0.009442 Unknown ETOH 0.34.826824 711.8344 24 H −2.25605 0.017007 Unknown ETOH 0.3 47.76987731.0954 4 D −3.25036 0.027339 Unknown ETOH 0.3 5.918027 732.007 4 D1.76125 0.038454 Unknown ETOH 0.3 35.028 751.4193 24 H −1.70279 0.045682Unknown ETOH 0.3 35.028 751.4193 4 D 1.845101 0.0375 Unknown ETOH 0.369.32865 765.5211 24 H 1.884393 0.027105 Unknown ETOH 0.3 34.33545774.4767 24 H −2.34161 0.036388 Unknown ETOH 0.3 34.60124 796.9917 4 D1.688919 0.047852 Unknown ETOH 0.3 4.613879 820.8181 24 H −1.851120.038895 Unknown ETOH 0.3 4.613879 820.8181 4 D −1.57113 0.014605Unknown ETOH 0.3 5.259716 888.8041 24 H 1.221624 0.043348 Unknown ETOH0.3 5.217833 913.8074 24 H −1.88465 0.026148 Unknown ETOH 0.3 5.399211921.0025 4 D 1.944905 2.78E−05 Unknown ETOH 0.3 5.387775 922.0048 24 H−2.75471 0.015691 Unknown ETOH 0.3 3.680188 980.075 4 D 1.4809260.012893 Unknown ETOH 0.3 3.646902 994.0917 4 D 1.604696 0.000395Unknown ETOH 0.3 3.705141 1008.072 4 D 1.415783 0.021503 Unknown ETOH0.3 5.177162 1038.786 4 D 1.588208 0.030971 Unknown ETOH 0.3 5.89051040.323 4 D −1.47887 0.009656 Unknown

All references cited herein are incorporated by reference. In addition,the invention is not intended to be limited to the disclosed embodimentsof the invention. It should be understood that the foregoing disclosureemphasizes certain specific embodiments of the invention and that allmodifications or alternatives equivalent thereto are within the spiritand scope of the invention as set forth in the appended claims.

1-54. (canceled)
 55. A biomarker profile for identifying a toxiccompound, wherein the biomarker profile comprises one or a plurality ofcellular metabolites having a molecular weight of from about 10 to about1500 Daltons that are differentially produced in human embryonic stemcells (hESCs) or hESC-derived lineage-specific cells contacted with atoxic compound or compounds, wherein the profile was identified from apopulation of cellular metabolites secreted from the cells.
 56. Abiomarker profile of claim 55, comprising tetrahydrofolate,dihydrofolate or other metabolites in the folate metabolic pathway,glutathione, or oxidized glutathione.
 57. A biomarker profile of claim55, comprising kynurenine, 8-methoxykynurenate, N′-formylkynurenine7,8-dihydro-7,8-dihydroxykynurenate 5-Hydroxytryptophan,N-acetyl-D-tryptophan, glutamate, pyroglutamic acid or other metabolitesin the tryptophan or glutamate metabolic pathways, histamine, dopamine,3,4-dihydroxybutyric acid, serotonin, gamma-aminobutyric acid (GABA) orother butyric acid species.